JP6964590B2 - Optically driven convection and displacement microfluidic devices, their kits and methods - Google Patents

Description

The present application is U.S. Provisional Patent Application No. 62 / 273,104 filed on December 30, 2015, U.S. Provisional Patent Application No. 62 / 314,889 filed on March 29, 2016, and 2016. It is a non-provisional application claiming interests under US Patent Law Article 119 (e) of US Provisional Patent Application No. 62 / 428,539 filed on December 1, and each of these provisional patent applications as a whole. Incorporated herein by reference.

Background As the field of microfluidics has evolved, microfluidic devices have become a convenient platform for the processing and manipulation of microscopic objects such as living cells. Some embodiments of the present invention relate to methods and devices that use optically driven air bubbles, convection, and displacement fluid flows to provide driving force in a microfluidic device.

Overview In one aspect, a microfluidic device is provided, the microfluidic device comprising an enclosure further including a flow region and an isolation enclosure, the isolation enclosure including a connection region, a separation region, and a displacement force generating region, the connection region. Includes a proximal opening to the flow region and a tip opening to the separation region, the separation region contains at least one fluid connection to the displacement force generation region, and the displacement force generation region further comprises a thermal target. ..

In another aspect, a microfluidic device is provided, the microfluidic device comprising an enclosure, the enclosure being a microfluidic circuit configured to include a fluid medium, wherein the microfluidic circuit is at least one of the fluid media. A microfluidic circuit configured to correspond to one circulation flow and a first thermal target located on the surface of the enclosure within the microfluidic circuit, the first thermal target being optically illuminated. It then includes a first thermal target configured to generate a first circulating flow of the fluid medium.

In yet another aspect, a microfluidic device is provided, the microfluidic device comprising an enclosure, the enclosure having a microfluidic channel and an isolation enclosure, in which the isolation enclosure is adjacent to the microfluidic channel and the microfluidic channel. Open in, a thermal target is placed in a channel adjacent to the opening to the isolation enclosure, and the thermal target is further configured to direct the flow of the fluid medium towards the isolation enclosure when optically illuminated.

In another aspect, a kit for culturing microobjects is provided, the kit being configured to provide the microfluidic device described herein and at least one covering surface within the enclosure of the microfluidic device. Includes one or more reagents.

In another aspect, a method of removing one or more micro-objects within a microfluidic device is provided, the method of which one or more micro-objects are placed in a fluid medium in the enclosure of the microfluidic device. A step of illuminating a selected discrete region containing or adjacent, the enclosure comprising a microfluidic circuit including a flow region and a substrate, a step of illuminating and a first time sufficient to generate a removal force. A step of maintaining illumination of a selected discrete region for a period of time, thereby including removing and maintaining one or more micro-objects from the surface.

In yet another aspect, a method of mixing the fluid medium and / or the micro-objects contained therein within the enclosure of the microfluidic device is provided, the method of which is a micro-fluid circuit containing at least one fluid medium and / or micro-objects. Focusing the light source on a thermal target located on the surface of the inner enclosure, thereby heating and focusing the first portion of at least one fluid medium and circulating the at least one fluid medium. Introducing a flow into a microfluidic circuit, thereby including mixing and introducing a fluid medium and / or microobjects arranged therein.

It is a schematic representation of a system used in combination with a microfluidic device and related control equipment according to some embodiments of the present disclosure. It is a graphic representation of a microfluidic device according to some embodiments of the present disclosure. It is a graphic representation of a microfluidic device according to some embodiments of the present disclosure. It is a graphic representation of a separation enclosure according to some embodiments of the present disclosure. It is a graphic representation of a separation enclosure according to some embodiments of the present disclosure. It is a graphic representation of a detailed isolation enclosure according to some embodiments of the present disclosure. It is a graphic representation of a quarantine enclosure according to some other embodiment of the present disclosure. It is a graphic representation of a quarantine enclosure according to some other embodiment of the present disclosure. It is a graphic representation of a quarantine enclosure according to some other embodiment of the present disclosure. It is a graphic representation of a microfluidic device according to the embodiment of the present disclosure. It is a diagram representation of the covering surface of the microfluidic device according to the embodiment of the present disclosure. FIG. 6 is a graphic representation of a particular example of a system used in conjunction with a microfluidic device and related control equipment according to some embodiments of the present disclosure. It is a schematic representation of an imaging device according to some embodiments of the present disclosure. It is a graphic representation of various thermal targets according to the embodiments of the present disclosure. It is a graphic representation of various thermal targets according to the embodiments of the present disclosure. It is a graphic representation of various thermal targets according to the embodiments of the present disclosure. It is a graphic representation of various thermal targets according to the embodiments of the present disclosure. It is a graphic representation of various thermal targets according to the embodiments of the present disclosure. It is a graphic representation of various thermal targets according to the embodiments of the present disclosure. It is a graphic representation of various thermal targets according to the embodiments of the present disclosure. It is a graphic representation of various thermal targets according to the embodiments of the present disclosure. It is a graphic representation of various thermal targets according to the embodiments of the present disclosure. It is a graphic representation of the isolation enclosure according to some embodiments of the present disclosure. It is a graphic representation of the isolation enclosure according to some embodiments of the present disclosure. It is a graphic representation of the isolation enclosure according to some embodiments of the present disclosure. It is a graphic representation of the isolation enclosure according to some embodiments of the present disclosure. It is a graphic representation of the isolation enclosure according to some embodiments of the present disclosure. It is a graphic representation of the isolation enclosure according to some embodiments of the present disclosure. It is a graphic representation of the isolation enclosure according to some embodiments of the present disclosure. It is a graphic representation of the isolation enclosure according to some embodiments of the present disclosure. It is a graphic representation of the isolation enclosure according to some embodiments of the present disclosure. It is a graphic representation of a further embodiment of the isolation enclosure according to the present disclosure. It is a graphic representation of a further embodiment of the isolation enclosure according to the present disclosure. It is a graphic representation of a further embodiment of the isolation enclosure according to the present disclosure. It is a graphic representation of a further embodiment of the isolation enclosure according to the present disclosure. It is a graphic representation of a further embodiment of the isolation enclosure according to the present disclosure. It is a graphic representation of a further embodiment of the isolation enclosure according to the present disclosure. A microfluidic device according to some embodiments of the present disclosure is shown. A microfluidic device according to some embodiments of the present disclosure is shown. A microfluidic device according to some embodiments of the present disclosure is shown. A microfluidic device according to some embodiments of the present disclosure is shown. A photographic representation of the use of optical driving forces used to eject cells from the isolation enclosure according to some embodiments of the present disclosure and subsequent viability. A photographic representation of the use of optical driving forces used to eject cells from the isolation enclosure according to some embodiments of the present disclosure and subsequent viability. A photographic representation of the use of optical driving forces used to eject cells from the isolation enclosure according to some embodiments of the present disclosure and subsequent viability. A photographic representation of the use of optical driving forces used to eject cells from the isolation enclosure according to some embodiments of the present disclosure and subsequent viability. The use of optically driven displacement to eject cells from the quarantine enclosure and subsequent viability is shown. The use of optically driven displacement to eject cells from the quarantine enclosure and subsequent viability is shown. The use of optically driven displacement to eject cells from the quarantine enclosure and subsequent viability is shown. It is a photographic representation of cells maintained in a microfluidic device before and after optical drive displacement according to the present disclosure. It is a photographic representation of cells maintained in a microfluidic device before and after optical drive displacement according to the present disclosure. It is a photographic representation of cells maintained in a microfluidic device before and after optical drive displacement according to the present disclosure. It is a photographic representation of cells maintained in a microfluidic device before and after optical drive displacement according to the present disclosure. It is a photographic representation of cells maintained in a microfluidic device before and after optical drive displacement according to the present disclosure. It is a photographic representation of cells maintained in a microfluidic device before and after optical drive displacement according to the present disclosure. It is a photographic representation of a method of using lighting to generate a circular flow in which a small object can move. It is a photographic representation of a method of using lighting to generate a circular flow in which a small object can move. It is a photographic representation of a method of using lighting to generate a circular flow in which a small object can move. It is a photographic representation of an embodiment of a laser illumination method for removing microscopic objects. It is a photographic representation of an embodiment of a laser illumination method for removing microscopic objects. It is a photographic representation of an embodiment of a laser illumination method for removing microscopic objects. It is a photographic representation of another embodiment of a method of laser illumination for removing microscopic objects. It is a photographic representation of another embodiment of a method of laser illumination for removing microscopic objects. It is a photographic representation of another embodiment of a method of laser illumination for removing microscopic objects. It is a photographic representation of another embodiment of a method of laser illumination for removing microscopic objects. It is a photographic representation of another embodiment of a method of laser illumination for removing microscopic objects.

Detailed Description of the exemplary Embodiments This specification describes exemplary embodiments and uses of the present disclosure. However, the present disclosure is not limited to these exemplary embodiments and uses or in the manner in which the exemplary embodiments and uses operate or are described herein. In addition, the figure may show a simplified or partial view, and the dimensions of the elements in the figure may not be emphasized or otherwise proportional. In addition, when the terms "above", "attached to", "connected to", "bonded to" or similar terms are used herein, certain elements (eg, materials, layers). , Substrates, etc.), whether or not one element is directly above another, directly attached, directly connected, or directly bonded, or between one element and another. It is "above" another element, with or without one or more intervening elements, and can "attach" to, "connect to", or "connect to" another element. .. Also, unless the context indicates otherwise, the direction (eg, up, down, top, bottom, side, top, bottom, bottom, top, top, bottom, horizontal, vertical, "x". , "Y", "z", etc.), when provided, are relative, provided by way of example only for ease of illustration and consideration, and not as a limitation. In addition, if a list of elements (eg, elements a, b, c) is mentioned, such a reference is any one of the elements listed in the list itself, less than all of the listed elements. And / or combinations of all the listed elements are intended to be included. Sectional divisions herein are for ease of consideration only and do not limit any combination of elements considered.

When the dimensions of a microfluidic feature are described as having width or area, the dimensions are usually both x-axis and / or y-axis dimensions in a plane parallel to the substrate and / or cover of the microfluidic device. Explained relative to. The height of the microfluidic features can be described relative to the z-axis direction orthogonal to the plane parallel to the substrate and / or cover of the microfluidic device. In some cases, the cross-sectional area of microfluidic features such as channels or passages may refer to the x-axis / z-axis, y-axis / z-axis, or x-axis / y-axis area.

As used herein, "substantially" means functioning well for the intended purpose. Therefore, the term "substantially" refers to small and insignificant variations from absolute or perfect conditions, dimensions, measurements, results, etc. as expected by one of ordinary skill in the art that do not significantly affect overall performance. Tolerate. When used with respect to a number, parameter, or characteristic that can be expressed as a number, "substantially" means within 10%.

The term "one" means more than one.

As used herein, the term "plurality" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more.

As used herein, the term "disposed" includes "located" within its meaning.

As used herein, a "microfluidic device" or "microfluidic device" is one or more separate microfluidic circuits configured to hold a fluid, each microfluidic circuit. A fluid (s) with one or more separate microfluidic circuits composed of fluidly interconnected circuit elements including, but not limited to, regions, channels, channels, chambers, and / or enclosures. And optionally at least one port configured to allow microobjects suspended in the fluid to flow into and / or out of the microfluidic device. Typically, the microfluidic circuit of a microfluidic device comprises a flow region that may include a microfluidic channel and at least one chamber and is less than about 1 mL, eg, less than about 750 μL, less than about 500 μL, less than about 250 μL, less than about 200 μL, about. Less than 150 μL, less than about 100 μL, less than about 75 μL, less than about 50 μL, less than about 25 μL, less than about 20 μL, less than about 15 μL, less than about 10 μL, less than about 9 μL, less than about 8 μL, less than about 7 μL, less than about 6 μL, less than about 5 μL Holds a volume of fluid less than about 4 μL, less than about 3 μL, or less than about 2 μL. In certain embodiments, the microfluidic circuit is about 1 μL to about 2 μL, about 1 μL to about 3 μL, about 1 μL to about 4 μL, about 1 μL to about 5 μL, about 2 μL to about 5 μL, about 2 μL to about 8 μL, about 2 μL to. About 10 μL, about 2 μL to about 12 μL, about 2 μL to about 15 μL, about 2 μL to about 20 μL, about 5 μL to about 20 μL, about 5 μL to about 30 μL, about 5 μL to about 40 μL, about 5 μL to about 50 μL, about 10 μL to about 50 μL , About 10 μL to about 75 μL, about 10 μL to about 100 μL, about 20 μL to about 100 μL, about 20 μL to about 150 μL, about 20 μL to about 200 μL, about 50 μL to about 200 μL, about 50 μL to about 250 μL, or about 50 μL to about 300 μL. Hold. The microfluidic circuit is fluid with a first end fluidly connected to a first port (eg, inlet) of the microfluidic device and a second port (eg, outlet) of the microfluidic device. It may be configured to have a second end connected to.

As used herein, "nanofluid device" or "nanofluid device" means less than about 1 μL, eg, less than about 750 nL, less than about 500 nL, less than about 250 nL, less than about 200 nL, less than about 150 nL, about. Less than 100 nL, less than about 75 nL, less than about 50 nL, less than about 25 nL, less than about 20 nL, less than about 15 nL, less than about 10 nL, less than about 9 nL, less than about 8 nL, less than about 7 nL, less than about 6 nL, less than about 5 nL, less than about 4 nL , A type of microfluidic device having a microfluidic circuit comprising at least one circuit element configured to hold a volume of fluid less than about 3 nL, less than about 2 nL, less than about 1 nL, or less. Nanofluid devices include multiple circuit elements (eg, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 15, 20, 25, 50). 75 pieces, 100 pieces, 150 pieces, 200 pieces, 250 pieces, 300 pieces, 400 pieces, 500 pieces, 600 pieces, 700 pieces, 800 pieces, 900 pieces, 1000 pieces, 1500 pieces, 2000 pieces, 2500 pieces, 3000 pieces, 3500 pieces, 4000 pieces, 4500 pieces, 5000 pieces, 6000 pieces, 7000 pieces, 8000 pieces, 9000 pieces, 10,000 pieces, or more) can be included. In certain embodiments, one or more (eg, all) of at least one circuit element is about 100 pL to about 1 nL, about 100 pL to about 2 nL, about 100 pL to about 5 nL, about 250 pL to about 2 nL, about. 250pL to about 5nL, about 250pL to about 10nL, about 500pL to about 5nL, about 500pL to about 10nL, about 500pL to about 15nL, about 750pL to about 10nL, about 750pL to about 15nL, about 750pL to about 20nL, about 1nL to It is configured to hold a fluid with a volume of about 10 nL, about 1 nL to about 15 nL, about 1 nL to about 20 nL, about 1 nL to about 25 nL, or about 1 nL to about 50 nL. In other embodiments, one or more (eg, all) of at least one circuit element is about 20 nL to about 200 nL, about 100 nL to about 200 nL, about 100 nL to about 300 nL, about 100 nL to about 400 nL, about 100 nL to about 400 nL. 100 nL to about 500 nL, about 200 nL to about 300 nL, about 200 nL to about 400 nL, about 200 nL to about 500 nL, about 200 nL to about 600 nL, about 200 nL to about 700 nL, about 250 nL to about 400 nL, about 250 nL to about 500 nL, about 250 nL to It is configured to hold a fluid with a volume of about 600 nL, or about 250 nL to about 750 nL.

As used herein, "microfluidic channel" or "flow channel" refers to the flow region of a microfluidic device that has a length substantially longer than both horizontal and vertical dimensions. For example, the flow channel is at least 5 times the length of either the horizontal or vertical dimension, eg, at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length. It can be times, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or longer. In some embodiments, the length of the flow channel is in the range of about 100,000 μm to about 500,000 μm, including any range in between. In some embodiments, the horizontal dimensions range from about 100 μm to about 1000 μm (eg, about 150 μm to about 500 μm) and the vertical dimensions range from about 25 μm to about 200 μm, such as from about 40 μm to about 150 μm. be. Note that flow channels can have a wide variety of different spatial configurations in microfluidic devices and are therefore not limited to perfectly linear elements. For example, the flow channel can be one or more parts having the following configurations: curves, curves, spirals, slopes, descents, branches (eg, different channels), and any combination thereof. Can include. In addition, the flow channels can have different cross-sectional areas along the path and can expand or contract to provide the desired fluid flow internally. The flow channel can include a valve, which can be of any type known in the field of microfluidics. Examples of microfluidic channels, including valves, are disclosed in US Pat. Nos. 6,408,878 and 9,227,200, each of which is incorporated herein by reference in its entirety. ..

As used herein, the term "obstacle" generally partially (but not completely) impedes the movement of a target microobject between two different regions or circuit elements within a microfluidic device. Refers to bumps large enough to be or similar types of structures. The two different regions / circuit elements can be, for example, the contiguous and isolated regions of the microfluidic isolation enclosure.

As used herein, the term "stenosis" generally refers to the narrowing of a circuit element (or interface between two circuit elements) within a microfluidic device. The stenosis can be located, for example, at the interface between the separation zone and the contiguous zone of the microfluidic isolation enclosure of the present disclosure.

As used herein, the term "transparent" refers to a material that, when visible light is transmitted, allows visible light to pass through substantially unchanged.

As used herein, the term "microscopic object" generally refers to any microscopic object that can be separated and / or manipulated by the present invention. Non-limiting examples of micro objects include fine particles; micro beads (eg, polystyrene beads, Luminex ™ beads, etc.); magnetic beads; micro rods; micro wires; inanimate micro objects such as quantum dots, cells; biology. Cellular organs; vesicles or complexes; synthetic vesicles; liposomes (eg, derived from synthetic or membrane specimens); biological microobjects such as lipid nanoraft, or inanimate microobjects and biological microobjects (For example, microbeads attached to cells, liposome-coated microbeads, liposome-coated magnetic beads, etc.) can be mentioned. The beads may contain covalent or non-covalent moieties / molecules such as fluorescent labels, proteins, carbohydrates, antigens, small molecule signaling moieties, or other chemical / biological species available in the assay. Lipid nanocrafts are described, for example, in Ritchie et al., (2009) "Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs", Methods Enzymol., 464: 211-231.

As used herein, the term "cell" is used synonymously with the term "living cell". Non-limiting examples of living cells include eukaryotic cells, plant cells, mammalian cells, reptile cells, avian cells, fish cells and other animal cells, prokaryotic cells, bacterial cells, fungal cells, progenitor cells and the like, muscle and cartilage. Cells dissociated from tissues such as tissues, fat, skin, liver, lungs, and nerve tissue, T cells, B cells, natural killer cells, immune cells such as macrophages, embryos (for example, mating cells), egg mother cells, eggs , Sperm cells, hybridomas, cultured cells, cells from cell lines, cancer cells, infected cells, transfected cells and / or transformed cells, reporter cells and the like. Mammalian cells can be, for example, cells from humans, mice, rats, horses, goats, sheep, cows, primates and the like.

A colony of living cells is a "clone" if all of the living cells within the reproductive colony are daughter cells derived from a single parent cell. In certain embodiments, all daughter cells within a clonal colony are from a single parent cell with less than 10 cell divisions. In other embodiments, all daughter cells within the clonal colony are from a single parent cell with 14 or less cell divisions. In other embodiments, all daughter cells within the clonal colony are from a single parent cell with 17 or less cell divisions. In other embodiments, all daughter cells within the clonal colony are from a single parent cell with less than 20 cell divisions. The term "cloned cell" refers to cells of the same clonal colony.

As used herein, a "colony" of living cells is two or more cells (eg, about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 8 to about. 80 cells, about 10 to about 100, about 20 about 200, about 40 about 400, about 60 about 600, about 80 about 800, about 100 about 1000, or more than about 1000 cells) ..

As used herein, the term "maintaining cells" is an optional choice as well as both fluid and gas components that provide the conditions necessary to keep cells alive and / or proliferate. Refers to providing an environment that includes the surface.

As used herein, the term "proliferation", when referring to cells, refers to an increase in cell number.

A "component" of a fluid medium is a medium comprising solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites and the like. Any chemical or biochemical molecule present in.

As used herein with reference to a fluid medium, "diffusing" and "diffusing" refer to the thermodynamic transfer of components of a fluid medium down a concentration gradient.

The phrase "medium flow" refers to the bulk movement of a fluid medium primarily due to any mechanism other than diffusion. For example, the flow of the medium can include the movement of the fluid medium from one point to another due to the pressure difference between the points. Such flows can include continuous flows of liquids, pulsed flows, periodic flows, random flows, intermittent flows, or reciprocating flows, or any combination thereof. When one fluid medium flows into another fluid medium, turbulence and mixing of the medium can occur.

The phrase "substantially no flow" refers to the flow rate of a fluid medium averaged over time that is less than the diffusivity of the components of the material (eg, sample of interest) into or in the fluid medium. .. The diffusivity of the components of such a material can depend, for example, on the temperature and size of the components and the strength of the interaction between the components and the fluid medium.

As used herein with reference to different regions within a microfluidic device, the phrase "fluidically connected" is used when the different regions are substantially filled with a fluid, such as a fluid medium. It means that the fluids in each region are connected to form a single fluid. This does not mean that the compositions of fluids (or fluid media) in different regions are necessarily the same. To be precise, fluids in different regions fluidly connected in a microfluidic device are fluid as the solute moves down each concentration gradient and / or the fluid flows through the microfluidic device. It can have different compositions (eg, different concentrations of solutes such as proteins, carbohydrates, ions, or other molecules).

As used herein, a "channel" is one or more fluidly connected circuit elements that define the trajectory of a medium flow and receive the trajectory of the medium flow (eg, a channel, etc.). Area, chamber, etc.). Therefore, the flow path is an example of a sweep area for a microfluidic device. Other circuit elements (eg, non-sweep regions) may be fluidly connected to circuit elements that include the flow path without being subject to the flow of media in the flow path.

As used herein, "separation of microobjects" means confining microobjects in a defined area within a microfluidic device. Microobjects may still be able to move within the in situ generated capture structure.

Microfluidic (or nanofluidic) devices can include "sweep" and "non-sweep" regions. As used herein, a "sweep" region is one or more fluidly interconnected circuit elements of a microfluidic circuit that receive the flow of a medium as the fluid flows through the microfluidic circuit. Consists of. The circuit elements of the sweep region can include, for example, all or part of the region, channel, and chamber. As used herein, a "non-sweep" region is one or more fluid interconnects of a microfluidic circuit that are substantially immune to fluid flow when the fluid is flowing through the microfluidic circuit. It is composed of the circuit elements. The non-sweep region may be fluidly connected to the sweep region if the fluid connection has a structure that allows diffusion between the sweep region and the non-sweep region but has virtually no medium flow. can. Thus, the microfluidic device has a structure that substantially separates the non-sweep region from the flow of media within the sweep region, while allowing substantially only diffusion flow between the sweep region and the non-sweep region. be able to. For example, the flow channel of a microfluidic device is an example of a sweep region, while the isolation region of a microfluidic device (discussed in more detail below) is an example of a non-sweep region.

As used herein, a "sacrificial feature" can be used as a thermal target in the microfluidic devices and methods of the present disclosure, producing bubbles, cavitation forces, or shear currents as described herein. Refers to a microfluidic circuit element that, when illuminated enough to do so, is at least partially destroyed.

In such microfluidic devices, the ability of biological microobjects (eg, living cells) to produce specific biological materials (eg, proteins such as antibodies) can be assayed. In certain embodiments of the assay, sample material containing biological micro-objects (eg, cells) assayed for the production of the sample of interest can be loaded into the sweep area of the microfluidic device. Each of the biological micro-objects (eg, mammalian cells such as human cells) can be selected for specific properties and can be placed in non-sweep regions. The remaining sample material can then be drained from the sweep region and the assay material can be flushed into the sweep region. Since the selected biological micro-object is in the non-sweep region, the selected biological micro-object is substantially unaffected by the outflow of the remaining sample material or the influx of assay material. The selected biological microobject can generate a sample of interest, the sample can diffuse from the non-sweep region into the sweep region, and in the sweep region, the specimen of interest. Can react with assay materials to generate localized detectable reactions, each of which can be correlated with a particular non-swept region. Analyze any non-swept area associated with the detected reaction to identify any of the biological micro-objects in the non-swept area that are sufficient producers of the sample of interest. can do.

Microfluidic devices and systems for operating and observing such devices Figure 1A can be used for embryo production in vitro, including selection and evaluation of eggs and / or oocytes and / or sperm. An example of the microfluidic device 100 and the system 150 is shown. A perspective view of the microfluidic device 100 is shown, in which the cover 110 is partially cut out and a partial view of the inside of the microfluidic device 100 is provided. The microfluidic device 100 generally includes a microfluidic circuit 120 that includes a flow path 106, through which the fluid medium 180 can flow, optionally one or more microobjects (not shown). ) Is conveyed within the microfluidic circuit 120 and / or through the microfluidic circuit 120. Although one microfluidic circuit 120 is shown in FIG. 1A, suitable microfluidic devices can include multiple (eg, 2 or 3) such microfluidic circuits. Regardless, the microfluidic device 100 can be configured to be a nanofluidic device. As shown in FIG. 1A, the microfluidic circuit 120 may include a plurality of microfluidic isolation enclosures 124, 126, 128, and 130, where each isolation enclosure is one or one fluidly connected to the flow path 106. It may have multiple openings. In some embodiments of the device of FIG. 1A, the isolation enclosure may have only one opening through the flow path 106. As further discussed below, the microfluidic isolation enclosure is optimal for holding microobjects in a microfluidic device, such as the microfluidic device 100, even when the medium 180 is flowing through the flow path 106. Includes various features and structures that have been modified. However, before referring to the above, an overview of the microfluidic device 100 and the system 150 is provided.

As generally shown in FIG. 1A, the microfluidic circuit 120 is defined by an enclosure 102. Although the enclosure 102 can be physically structured in different configurations, in the example shown in FIG. 1A, the enclosure 102 includes a support structure 104 (eg, a base), a microfluidic circuit structure 108, and a cover 110. It is shown as a thing. The support structure 104, the microfluidic circuit structure 108, and the cover 110 can be attached to each other. For example, the microfluidic circuit structure 108 can be arranged on the inner surface 109 of the support structure 104, and the cover 110 can be arranged so as to cover the microfluidic circuit structure 108. Together with the support structure 104 and the cover 110, the microfluidic circuit structure 108 can define the elements of the microfluidic circuit 120.

As shown in FIG. 1A, the support structure 104 may be at the bottom of the microfluidic circuit 120 and the cover 110 may be at the top of the microfluidic circuit 120. Alternatively, the support structure 104 and cover 110 may be configured in other orientations. For example, the support structure 104 may be at the top of the microfluidic circuit 120 and the cover 110 may be at the bottom of the microfluidic circuit 120. Regardless, there may be one or more ports 107, each containing a passage into or out of the enclosure 102. Examples of passages include valves, gates, through holes and the like. As shown, port 107 is a through hole created by a gap in the microfluidic circuit structure 108. However, the port 107 can be located in other components of the enclosure 102, such as the cover 110. Although only one port 107 is shown in FIG. 1A, the microfluidic circuit 120 can have two or more ports 107. For example, there may be a first port 107 that acts as an inlet for the fluid to enter the microfluidic circuit 120 and a second port 107 that acts as an outlet for the fluid to exit the microfluidic circuit 120. Whether the port 107 functions as an inlet or an outlet may depend on the direction in which the fluid flows through the flow path 106.

The support structure 104 can include one or more electrodes (not shown) and a substrate or a plurality of interconnected substrates. For example, the support structure 104 can include one or more semiconductor substrates, each semiconductor substrate being electrically connected to an electrode (eg, all or a subset of semiconductor substrates are electrically connected to one electrode. Can be connected to). The support structure 104 may further include a printed circuit board assembly (“PCBA”). For example, the semiconductor substrate can be mounted on the PCBA.

The microfluidic circuit structure 108 can define the circuit elements of the microfluidic circuit 120. Such circuit elements may include flow regions (which may include one or more flow channels), chambers, enclosures, traps, etc., which can be fluidly interconnected when the microfluidic circuit 120 is filled with fluid. Space or area of. In the microfluidic circuit 120 shown in FIG. 1A, the microfluidic circuit 108 includes a frame 114 and a microfluidic circuit material 116. The frame 114 can partially or completely enclose the microfluidic circuit material 116. The frame 114 can be, for example, a relatively rigid structure that substantially surrounds the microfluidic circuit material 116. For example, the frame 114 can include a metallic material.

In the microfluidic circuit material 116, cavities and the like can be patterned to define circuit elements and interconnections of the microfluidic circuit 120. The microfluidic circuit material 116 can include flexible materials such as flexible polymers that may be gas permeable (eg, rubber, plastics, elastomers, silicones, polydimethylsiloxane (“PDMS”), etc.). Other examples of materials that can constitute microfluidic circuit material 116 include molded glass, etchable materials such as silicone (photopatternable silicone or "PPS"), photoresists (eg, SU8), and the like. .. In some embodiments, such a material-thus the microfluidic circuit material 116-can be substantially impermeable to stiffness and / or gas. Regardless, the microfluidic circuit material 116 can be placed on the support structure 104 and inside the frame 114.

The cover 110 can be an integral part of the frame 114 and / or the microfluidic circuit material 116. Alternatively, the cover 110 can be a structurally distinct element, as shown in FIG. 1A. The cover 110 may contain the same or different material as the frame 114 and / or the microfluidic circuit material 116. Similarly, the support structure 104 may have a structure separate from the frame 114 or the microfluidic circuit material 116 as shown, or may be an integral part of the frame 114 or the microfluidic circuit material 116. Similarly, the frame 114 and the microfluidic circuit material 116 may have separate structures as shown in FIG. 1A, or may be integral parts of the same structure.

In some embodiments, the cover 110 may include a rigid material. The rigid material can be glass or a material with similar properties. In some embodiments, the cover 110 may include a deformable material. The deformable material can be a polymer such as PDMS. In some embodiments, the cover 110 can include both rigid and deformable materials. For example, one or more parts of cover 110 (eg, one or more parts located on isolation enclosures 124, 126, 128, 130) include deformable materials that interface with the rigid material of cover 110. be able to. In some embodiments, the cover 110 may further include one or more electrodes. One or more electrodes can include conductive oxides such as indium-tin-oxide (ITO) that can be coated with glass or similar insulating material. Alternatively, one or more electrodes may be monolayer nanotubes, multilayer nanotubes, nanowires, clusters of conductive nanoparticles, or a combination thereof, embedded in a deformable polymer such as a polymer (eg, PDMS). It can be a flexible electrode. Flexible electrodes that can be used in microfluidic devices are described, for example, in US Patent Application Publication No. 2012/0325665 (Chiou et al.), The contents of which are incorporated herein by reference. In some embodiments, the cover 110 can be modified to support cell adhesion, survival, and / or growth (eg, all or part of a surface facing inward towards the microfluidic circuit 120). By adjusting). Modifications may include coatings of synthetic or natural polymers. In some embodiments, the cover 110 and / or the support structure 104 is capable of transmitting light. The cover 110 may also contain at least one gas permeable material (eg, PDMS or PPS).

FIG. 1A also shows a system 150 that operates and controls a microfluidic device such as the microfluidic device 100. The system 150 includes a power supply 192, an imaging device 194 (embedded in the imaging module 164, the device 194 itself is not shown in FIG. 1A), and a tilting device 190 (part of the tilting module 166, where the device 190 is FIG. 1A. Not shown in).

The power supply 192 can provide power to the microfluidic device 100 and / or tilt device 190 and provide a bias voltage or current as needed. The power supply 192 can include, for example, one or more alternating current (AC) and / or direct current (DC) voltage sources or current sources. The imaging device 194 (part of the imaging module 164 described below) can include a device such as a digital camera that captures an image inside the microfluidic circuit 120. In some cases, the imaging device 194 further includes a detector with a high frame rate and / or high sensitivity (eg, for low light applications). The imaging device 194 directs the stimulating radiation and / or the light beam into the microfluidic circuit 120, and the radiation and / or emitted from the microfluidic circuit 120 (or a small object contained in the microfluidic circuit 120). It can also include a mechanism for collecting light rays. The emitted light can be within the visible spectrum and may include, for example, fluorescent radiation. The reflected light may include reflected radiation emitted from a wide spectrum lamp such as an LED or a mercury lamp (eg, a high pressure mercury lamp) or a xenon arc lamp. As discussed with respect to FIG. 3B, the imaging device 194 may further include a microscope (or optical column), which may or may not include an eyepiece.

The system 150 further includes a tilt device 190 (part of the tilt module 166 described below) configured to rotate the microfluidic device 100 around one or more axes of rotation. In some embodiments, the tilt device 190 orients the microfluidic device 100 (and thus the microfluidic circuit 120) horizontally (ie, 0 ° relative to the x-axis and y-axis) and vertically (ie, x-axis). And / or 90 ° with respect to the y-axis), or support and / or support the enclosure 102 containing the microfluidic circuit 120 around at least one axis so that it can be held in any orientation between them. Configured to hold. The orientation of the microfluidic device 100 (and the microfluidic circuit 120) relative to the axis is referred to herein as the "tilt" of the microfluidic device 100 (and the microfluidic circuit 120). For example, the tilt device 190 is 0.1 °, 0.2 °, 0.3 °, 0.4 °, 0.5 °, 0.6 °, 0.7 °, 0. 8 °, 0.9 °, 1 °, 2 °, 3 °, 4 °, 5 °, 10 °, 15 °, 20 °, 25 °, 30 °, 35 °, 40 °, 45 °, 50 °, The microfluidic device 100 can be tilted at 55 °, 60 °, 65 °, 70 °, 75 °, 80 °, 90 °, or any degree in between. The horizontal orientation (and therefore the x-axis and y-axis) is defined as perpendicular to the vertical axis defined by gravity. The tilting device tilts the microfluidic device 100 (and the microfluidic circuit 120) at any angle greater than 90 ° relative to the x-axis and / or the y-axis, or the microfluidic device 100 (and the microfluidic circuit 120). The microfluidic device 100 (and the microfluidic circuit 120) can also be reversed by tilting 120) 180 ° relative to the x-axis or y-axis. Similarly, in some embodiments, the tilt device 190 tilts the microfluidic device 100 (and the microfluidic circuit 120) around a axis of rotation defined by the flow path 106 or any other part of the microfluidic circuit 120. Let me.

In some cases, the microfluidic device 100 is vertically tilted so that the flow path 106 is located above or below one or more isolation enclosures. The term "upper", as used herein, means that the flow path 106 is located above one or more isolation enclosures on the vertical axis defined by gravity (ie, above the flow path 106). The object in the isolation enclosure has higher gravitational potential energy than the object in the flow path). As used herein, the term "downward" means that the flow path 106 is located below one or more isolation enclosures on the vertical axis defined by gravity (ie, of the flow path 106). Indicates that the object in the lower isolation enclosure has lower gravitational potential energy than the object in the flow path).

In some cases, the tilt device 190 tilts the microfluidic device 100 around an axis parallel to the flow path 106. In addition, the microfluidic device 100 is at an angle of less than 90 ° so that the flow path 106 is located above or below the isolation enclosure rather than directly above or below the isolation enclosure. Can be tilted. In other cases, the tilt device 190 tilts the microfluidic device 100 around an axis orthogonal to the flow path 106. In yet other cases, the tilt device 190 tilts the microfluidic device 100 around an axis that is neither parallel nor orthogonal to the flow path 106.

System 150 can further include medium source 178. The medium source 178 (eg, container, reservoir, etc.) can include multiple sections or containers, each holding a different fluid medium 180. Therefore, the medium source 178 can be a device that is external to the microfluidic device 100 and is separate from the microfluidic device 100, as shown in FIG. 1A. Alternatively, the medium source 178 can be placed, in whole or in part, inside the enclosure 102 of the microfluidic device 100. For example, medium source 178 can include a reservoir that is part of the microfluidic device 100.

FIG. 1A also shows a simplified block diagram representation of an example of a control and monitoring device 152 that constitutes a portion of the system 150 and can be used in conjunction with the microfluidic device 100. As shown, an example of such a control and monitoring device 152 is a medium module 160 controlling the medium source 178 and a micro-object (not shown) and / or medium (eg, medium) in the microfluidic circuit 120. A prime mover module 162 that controls the movement and / or selection of droplets) and an imaging device that captures an image (eg, a digital image) 194 (eg, a camera, microscope, light source, or any combination thereof). Includes a master controller 154 that includes a module 164 and a tilt module 166 that controls the tilt device 190. The control device 152 may also include other modules 168 that control, monitor, or perform other functions relating to the microfluidic device 100. As shown, the device 152 can further include a display device 170 and an input / output device 172.

The master controller 154 can include a control module 156 and a digital memory 158. The control module 156 may include, for example, a digital processor configured to operate according to machine executable instructions (eg, software, firmware, source code, etc.) stored as non-temporary data or signals in memory 158. can. Alternatively or additionally, the control module 156 can include hard-wired digital and / or analog circuits. The medium module 160, the prime mover module 162, the imaging module 164, the tilt module 166, and / or the other module 168 can be configured similarly. Thus, a function, process, action, operation, or process step considered herein as being performed with respect to the microfluidic device 100 or any other microfluidic device is a master controller configured as described above. It can be performed by any one or more of 154, the medium module 160, the prime mover module 162, the imaging module 164, the tilt module 166, and / or the other module 168. Similarly, the master controller 154, medium module 160, prime mover module 162, imaging module 164, tilt module 166, and / or other modules 168 are communicably coupled to any function discussed herein. It can send and receive data used in processes, actions, actions, or steps.

The medium module 160 controls the medium source 178. For example, the medium module 160 can control the medium source 178 to put the selected fluid medium 180 into the enclosure 102 (eg, via the inlet 107). The medium module 160 can also control the removal of medium from the enclosure 102 (eg, through an outlet (not shown)). Therefore, one or more media can be selectively put into the microfluidic circuit 120 and carried out of the microfluidic circuit 120. The medium module 160 can also control the flow of the fluid medium 180 in the flow path 106 inside the microfluidic circuit 120. For example, in some embodiments, the medium module 160 is the flow of medium 180 through the flow path 106 and through the enclosure 102 before the tilt module 166 tilts the microfluidic device 100 to the tilt device 190 to the desired tilt angle. To stop.

The prime mover module 162 may be configured to control the selection, capture, and movement of microobjects (not shown) in the microfluidic circuit 120. As will be described later with respect to FIGS. 1B and 1C, the enclosure 102 may include a dielectrophoresis (DEP) configuration, photoelectron tweezers (OET) configuration, and / or photoelectron wetting (OEW) configuration (not shown in FIG. 1A). The prime mover module 162 can control the activation of electrodes and / or transistors (eg, tweezers) and micro-objects (not shown) in flow paths 106 and / or isolation enclosures 124, 126, 128, 130. / Or droplets of medium (not shown) can be selected and moved.

The image pickup module 164 can control the image pickup device 194. For example, the image pickup module 164 can receive and process image data from the image pickup device 194. The image data from the imaging device 194 can include any type of information captured by the imaging device 194 (eg, the presence or absence of accumulation of labels such as microscopic objects, medium droplets, fluorescent labels, etc.). Using the information captured by the imaging device 194, the imaging module 164 further increases the location of objects (eg, microobjects, droplets of medium) and / or the speed of movement of such objects within the microfluidic device 100. Can be calculated.

The tilt module 166 can control the tilt movement of the tilt device 190. Alternatively or additionally, the tilt module 166 can control the tilt ratio and timing to optimize the transfer of micro-objects to one or more isolation enclosures via gravity. The tilt module 166 is communicably coupled with the imaging module 164 to receive data describing the movement of microobjects and / or medium droplets in the microfluidic circuit 120. Using this data, the tilt module 166 can adjust the tilt of the microfluidic circuit 120 to adjust the rate at which microobjects and / or medium droplets move within the microfluidic circuit 120. The tilt module 166 can also use this data to iteratively adjust the position of microobjects and / or medium droplets within the microfluidic circuit 120.

In the example shown in FIG. 1A, the microfluidic circuit 120 is shown to include a microfluidic channel 122 and isolation enclosures 124, 126, 128, 130. Each enclosure includes an opening to the channel 122, but the enclosure may substantially separate the micro-objects within the enclosure from the fluid medium 180 and / or the channel 106 of channel 122 or other micro-objects within the enclosure. Others are closed so that you can. The walls of the isolation enclosure extend from the inner surface 109 of the base to the inner surface of the cover 110 to provide the enclosure. The opening of the enclosure to the microfluidic channel 122 is tilted with respect to the flow 106 so that the flow 106 is not directed into the enclosure. The flow can be tangential or orthogonal to the plane of the enclosure opening. In some cases, enclosures 124, 126, 128, 130 are configured to physically enclose one or more microobjects within the microfluidic circuit 120. Isolation enclosures according to the present disclosure may include various shapes, surfaces, and features optimized for use with DEP, OET, OEW, fluid flow, and / or gravity, as discussed in detail below. can.

The microfluidic circuit 120 may include any number of microfluidic isolation enclosures. Although five isolation enclosures are shown, the microfluidic circuit 120 may have fewer or more isolation enclosures. As shown, the microfluidic isolation enclosures 124, 126, 128, and 130 of the microfluidic circuit 120 provide one or more advantages useful in producing embryos, such as the separation of an egg from an adjacent egg. Each contains different features and shapes to be obtained. Testing, simulation, and fertilization can all be performed on an individual basis, and in some embodiments, on an individual timescale. In some embodiments, the microfluidic circuit 120 comprises a plurality of identical microfluidic isolation enclosures.

In some embodiments, the microfluidic circuit 120 comprises a plurality of microfluidic isolation enclosures, in which case two or more of the isolation enclosures contain different structures and / or features that provide different advantages in embryonic development. .. As a non-limiting example, keeping an egg in one type of enclosure while maintaining sperm in a different type of enclosure, in another embodiment, at least one of the isolation enclosures. It is configured to have electrical contacts suitable for providing electrical activation to the egg. In yet another embodiment, PEG (intervening) cells from different types of cells (eg, uterine cells, endometrial cells, uterine tubes (eg, oviduct or fallopian tubes), etc.) , Cumulus cells, or combinations thereof) can be placed in an enclosure adjacent to the enclosure containing the eggs, whereby secretions from the surrounding enclosures exit each enclosure and contain the eggs. It can diffuse into, which is not possible with in vitro culture and fertilization on a macro scale. Microfluidic devices useful for embryo development may include isolation enclosures 124, 126, 128, and 130 or any of their variants, and / or, as described below, FIGS. 2B, 2C, 2D, It may include an enclosure configured as shown in FIGS. 2E and 2F.

In the embodiment shown in FIG. 1A, one channel 122 and one channel 106 are shown. However, other embodiments may include a plurality of channels 122, each configured to include a flow path 106. The microfluidic circuit 120 further includes an inflow valve or port 107 for fluid communication with the flow path 106 and the fluid medium 180, whereby the fluid medium 180 can access the channel 122 through the inflow port 107. In some cases, the flow path 106 includes one path. In some cases, one path is arranged in a zigzag pattern, whereby the flow path 106 travels over the microfluidic device 100 more than once in alternating directions.

In some cases, the microfluidic circuit 120 includes a plurality of parallel channels 122 and channels 106, with the fluid medium 180 in each channel 106 flowing in the same direction. In some cases, the fluid medium in each channel 106 flows in at least one of the forward and reverse directions. In some cases, the quarantine enclosures are configured so that the quarantine enclosures can be placed in parallel with the target micro-objects (eg, relative to channel 122).

In some embodiments, the microfluidic circuit 120 further comprises one or more microobject traps 132. The trap 132 is generally formed on the wall forming the boundary of the channel 122 and may be located opposite one or more openings of the microfluidic isolation enclosures 124, 126, 128, 130. In some embodiments, the trap 132 is configured to receive or capture a small object from the flow path 106. In some embodiments, the trap 132 is configured to receive or capture a plurality of microobjects from the flow path 106. In some cases, the trap 132 contains a volume approximately equal to the volume of one target microobject.

The trap 132 may further include an opening configured to assist the flow of the target microobject to the trap 132. In some cases, the trap 132 includes an opening having a height and width approximately equal to the dimensions of one target micro-object, thereby preventing larger micro-objects from entering the micro-object trap. The trap 132 may further include other features configured to assist in the retention of the target micro-object within the trap 132. In some cases, the trap 132 is aligned with the opening of the microfluidic isolation enclosure and is located opposite the channel 122 with respect to the opening of the microfluidic isolation enclosure, thereby the axis parallel to the microfluidic channel 122. When the microfluidic device 100 is tilted around, the captured micro-object exits the trap 132 in a trajectory that drops the micro-object into the opening of the isolation enclosure. In some cases, the trap 132 includes a side passage 134 that is smaller than the target micro-object and facilitates flow through the trap 132, thereby increasing the probability of capturing the micro-object into the trap 132.

In some embodiments, dielectrophoretic (DEP) forces are applied over the fluid medium 180 via one or more electrodes (not shown) (eg, in channels and / or isolation enclosures) and internally. Manipulates, transports, separates, and sorts micro-objects placed in. For example, in some embodiments, the DEP force is applied to one or more parts of the microfluidic circuit 120 to transport one microobject from the flow path 106 to the desired microfluidic isolation enclosure. In some embodiments, DEP forces are used to prevent microscopic objects within the isolation enclosure (eg, isolation enclosure 124, 126, 128, or 130) from being displaced from the isolation enclosure. In addition, in some embodiments, DEP forces are used to selectively remove micro-objects previously collected according to the embodiments of the present disclosure from the isolation enclosure. In some embodiments, the DEP force includes optoelectronic tweezers (OET) force.

In other embodiments, a photoelectron wetting (OEW) force is applied to one or more of the support structures 104 (and / or cover 110) of the microfluidic device 100 via one or more electrodes (not shown). It is applied to multiple positions (eg, positions that help define channels and / or isolation enclosures) to manipulate, transport, separate, and sort droplets placed in the microfluidic circuit 120. For example, in some embodiments, OEW forces are applied to one or more locations on the support structure 104 (and / or cover 110) to allow one droplet from the flow path 106 to a desired microfluidic isolation enclosure. To transport to. In some embodiments, OEW forces are used to ensure that droplets within the isolation enclosure (eg, isolation enclosure 124, 126, 128, or 130) do not displace from the isolation enclosure. In addition, in some embodiments, OEW forces are used to selectively remove droplets previously collected according to the embodiments of the present disclosure from the isolation enclosure.

In some embodiments, the DEP and / or OEW forces are combined with other forces such as flow and / or gravity to manipulate, transport, and separate micro-objects and / or droplets within the microfluidic circuit 120. , And sort. For example, the enclosure 102 can be tilted (eg, by a tilting device 190) to position the flow path 106 and the micro-objects located within the flow path 106 over the microfluidic isolation enclosure, and gravity is the micro-object. And / or droplets can be transported within the enclosure. In some embodiments, the DEP force and / or the OEW force can be applied before other forces. In other embodiments, the DEP force and / or the OEW force can be applied after the other force. In yet other cases, the DEP force and / or the OEW force can be applied simultaneously with or alternately with the other forces.

1B, 1C and 2A-2H show various embodiments of microfluidic devices that can be used in the embodiments of the present disclosure. FIG. 1B shows an embodiment in which the microfluidic device 200 is configured as an optically actuated electrokinetic device. Various optically actuated electrokinetic devices are known in the art, including devices with optoelectronic tweezers (OET) configurations and devices with optoelectronic wetting (OEW) configurations. Examples of suitable OET configurations are set forth in the following U.S. Patent Documents, each of which is incorporated herein by reference in its entirety: U.S. Pat. No. RE44,711 (Wu et al.) (Originally U.S. Patent). (Issued as No. 7,612,355); and U.S. Pat. No. 7,965,339 (Ohta et al.). Examples of OEW configurations are US Pat. No. 6,958,132 (Chiou et al.) And US Patent Application Publication No. 2012/0024.
Shown in No. 708 (Chiou et al.), Both of which are incorporated herein by reference in their entirety. Yet another example of an optically actuated electrokinetic device includes an OET / OEW coupling configuration, examples of which are US Patent Application Publication Nos. 20150306598 (Khandros et al.) And 20150306599 (Khandros et al.) And their corresponding counterparts. It is set forth in the PCT publications International Publication No. 2015/1644846 and International Publication No. 2015/1644847, all of which are incorporated herein by reference in their entirety.

Examples of microfluidic devices with isolation enclosures capable of placing, culturing, and / or monitoring egg mother cells, eggs, or embryos include, for example, US Patent Application Publication No. 2014/0116881 (Application No. 14 /). 060, 117, filed October 22, 2013), US Patent Application Publication No. 2015/0151298 (Application Nos. 14/520, 568, filed October 22, 2014), and US Patent Application Publication No. 2015/015436. (Application No. 14 / 521,447, filed October 22, 2014), each of which is incorporated by reference in its entirety. U.S. Patent Application Publication Nos. 14 / 520,568 and 14 / 521,447 also describe exemplary methods for analyzing the secretions of cells cultured in microfluidic devices. Each of the above applications further describes a microfluidic device configured to generate a dielectrophoretic (DEP) force, such as a photoelectron tweezers (OET), or to provide photoelectron wetting (OEW). doing. For example, the photoelectron tweezers device shown in FIG. 2 of US Patent Application Publication No. 2014/0116881 is utilized in embodiments of the present disclosure to select individual biological micro-objects or groups of biological micro-objects. This is an example of a device that can be moved.

Primary Microfluidic Device Configuration As described above, system control and monitoring equipment can include a prime mover module that selects and moves objects such as micro-objects or droplets in the microfluidic circuit of the microfluidic device. Microfluidic devices can have different prime mover configurations depending on the type of object being moved and other considerations. For example, a dielectrophoresis (DEP) configuration can be used to select and move micro-objects in a microfluidic circuit. Thus, the support structure 104 and / or cover 110 of the microfluidic device 100 selectively induces a DEP force on the microobjects in the fluid medium 180 in the microfluidic circuit 120, thereby the individual microobjects or It can include a DEP configuration that selects, captures, and / or moves a group of microobjects. Alternatively, the support structure 104 and / or cover 110 of the microfluidic device 100 selectively induces an electron wetting (EW) force on the droplets in the fluid medium 180 in the microfluidic circuit 120. It can include an electronic wetting (EW) configuration that selects, captures, and / or moves individual droplets or droplet groups.

An example of the microfluidic device 200 including the DEP configuration is shown in FIGS. 21B and 1C. For brevity, FIGS. 1B and 1C show side and top cross sections of a portion of the enclosure 102 of the microfluidic device 200 having an open region / chamber 202, respectively, where the region / chamber 202 is the growth chamber. It should be understood that it can be part of a fluid circuit element having a more detailed structure, such as an isolation enclosure, a flow region, or a flow channel. In addition, the microfluidic device 200 may include other fluid circuit elements. For example, the microfluidic device 200 can include multiple growth chambers, such as those described herein for the microfluidic device 100, or isolation enclosures and / or one or more flow regions or flow channels. The DEP configuration can be incorporated into or selected from any such fluid circuit element of the microfluidic device 200. It should be further understood that any of the above or below microfluidic device and system components can be incorporated within the microfluidic device 200 and / or used in combination with the microfluidic device 200. For example, a system 150 including the above-mentioned control and monitoring device 152 including one or more of a medium module 160, a prime mover module 162, an imaging module 164, a tilt module 166, and another module 168 is used in combination with a microfluidic device 200. obtain.

As seen in FIG. 1B, the microfluidic device 200 includes a support structure 104 having an electrode activating substrate 206 overlapping the lower electrode 204 and the lower electrode 204, and a cover 110 having the upper electrode 210, the upper electrode 210. Is separated from the lower electrode 204. The upper electrode 210 and the electrode activating substrate 206 define both sides of the region / chamber 202. Therefore, the medium 180 contained in the region / chamber 202 provides a resistance connection between the upper electrode 210 and the electrode activating substrate 206. Also shown is a power supply 212 that is connected between the lower electrode 204 and the upper electrode 210 and is configured to generate a bias voltage between the electrodes as needed for the generation of DEP force in the region / chamber 202. There is. The power supply 212 can be, for example, an alternating current (AC) power supply.

In certain embodiments, the microfluidic device 200 shown in FIGS. 1B and 1C can have an optically actuated DEP configuration. Therefore, the change pattern of the light 218 from the light source 216, which can be controlled by the prime mover module 162, can selectively activate or deactivate the change pattern of the DEP electrode in the region 214 of the inner surface 208 of the electrode activation substrate 206. can. (Hereinafter, the region 214 of the microfluidic device having the DEP configuration is referred to as a "DEP electrode region"). As shown in FIG. 1C, the light pattern 218 directed at the inner surface 208 of the electrode activating substrate 206 can illuminate the selected DEP electrode region 214a (shown in white) with a pattern such as a square. The unilluminated DEP electrode region 214 (shaded) is hereinafter referred to as the "dark" DEP electrode region 214. The relative electrical impedance through the DEP electrode activation substrate 206 (ie, from the lower electrode 204 to the inner surface 208 of the electrode activation substrate 206 interfaceing with the medium 180 in the flow region 106) is the region in each dark DEP electrode region 214. It is greater than the relative electrical impedance through the medium 180 in the / chamber 202 (ie, from the inner surface 208 of the electrode activating substrate 206 to the upper electrode 210 of the cover 110). However, the illuminated DEP electrode region 214a exhibits a reduction in relative impedance through the electrode activated substrate 206 that is less than the relative impedance through the medium 180 in the region / chamber 202 in each illuminated DEP electrode region 214a.

When the power supply 212 is activated, the above DEP configuration creates an electric field gradient in the fluid medium 180 between the illuminated DEP electrode region 214a and the adjacent dark DEP electrode region 214, and then the electric field gradient is , Generates a local DEP force that attracts or rejects nearby microscopic objects (not shown) in the fluid medium 180. Therefore, the DEP electrode that attracts or rejects microscopic objects in the fluid medium 180 often by changing the light pattern 218 projected from the light source 216 to the microfluidic device 200, at the inner surface 208 of the region / chamber 202. It can be selectively activated and deactivated in different such DEP electrode regions 214. Whether the DEP force attracts or rejects nearby micro-objects may depend on parameters such as the frequency of the power supply 212 and the dielectric properties of the medium 180 and / or micro-objects (not shown).

The square pattern 220 of the illuminated DEP electrode region 214a shown in FIG. 1C is merely an example. The light pattern 218 projected onto the microfluidic device 200 can illuminate (activate) any pattern of the DEP electrode region 214, and the illuminated / activated pattern of the DEP electrode region 214 is light. It can be changed repeatedly by changing or moving the pattern 218.

In some embodiments, the electrode activating substrate 206 comprises or can be made of a photoconducting material. In such an embodiment, the inner surface 208 of the electrode activated substrate 206 may have no features. For example, the electrode activating substrate 206 may include or consist of a layer of hydrogenated amorphous silicon (a—Si: H) or a layer of a—Si: H. a-Si: H can contain, for example, about 8% to 40% hydrogen (calculated as the number of hydrogen atoms / the total number of hydrogen and silicon atoms multiplied by 100). The layer a—Si: H can have a thickness of about 500 nm to about 2.0 μm. In such an embodiment, the DEP electrode region 214 can be created in any pattern at any location on the inner surface 208 of the electrode activation substrate 206 by the optical pattern 218. Therefore, the number and pattern of the DEP electrode regions 214 need not be fixed and can correspond to the optical pattern 218. An example of a microfluidic device having a DEP configuration including a photoconductive layer as described above is, for example, US Pat. No. RE44,711 (Wu et al.) (Originally issued as US Pat. No. 7,612,355). Incorporated herein by reference in its entirety.

In another embodiment, the electrode activating substrate 206 can include a substrate including a plurality of dope layers, insulating layers (or regions), and conductive layers that form semiconductor integrated circuits known in the semiconductor field. For example, the electrode activation substrate 206 can include, for example, a plurality of phototransistors including a horizontal bipolar phototransistor, and each phototransistor corresponds to a DEP electrode region 214. Alternatively, the electrode activation substrate 206 can include electrodes controlled by a phototransistor switch (eg, conductive metal electrodes), each such electrode corresponding to the DEP electrode region 214. The electrode activation substrate 206 can include such a patterned phototransistor or phototransistor-controlled electrode. The pattern can be a substantially square phototransistor or an array of phototransistor-controlled electrodes arranged in a matrix, for example as shown in FIG. 2B. Alternatively, the pattern can be a substantially hexagonal phototransistor forming a hexagonal lattice or an array of phototransistor-controlled electrodes. Regardless of the pattern, the electrical circuit element can form an electrical connection between the DEP electrode region 214 and the lower electrode 210 on the inner surface 208 of the electrode activating substrate 206 and their electrical connection (ie, phototransistor or The electrode) can be selectively activated or deactivated by the light pattern 218. If not activated, each electrical connection has a relative impedance through the electrode activating substrate 206 (ie, from the lower electrode 204 to the inner surface 208 of the electrode activating electrode 206 interfaceing with the medium 180 in the region / chamber 202). It can have a high impedance that is greater than the relative impedance through the medium 180 in the corresponding DEP electrode region 214 (ie, from the inner surface 208 of the electrode activating substrate 206 to the upper electrode 210 of the cover 110). However, when activated by light within the light pattern 218, the relative impedance through the electrode activation substrate 206 is less than the relative impedance through the medium 180 in each illuminated DEP electrode region 214, thereby as described above. In addition, it activates the DEP electrode in the corresponding DEP electrode region 214. Thus, the DEP electrodes that attract or reject microscopic objects (not shown) in the medium 180 are many on the inner surface 208 of the electrode activation substrate 206 in the region / chamber 202, as determined by the light pattern 218. It can be selectively activated and deactivated in different DEP electrode regions 214.

An example of a microfluidic device having an electrode activating substrate containing a phototransistor is described, for example, in US Pat. No. 7,965,339 (Ohta et al.) (Eg, device 300 shown in FIGS. 21 and 22). Also see its description), the entire contents of which are incorporated herein by reference. Examples of microfluidic devices having electrode activation substrates that include electrodes controlled by phototransistor switches are described, for example, in US Patent Application Publication No. 2014/0124370 (Short et al.) (Eg, shown throughout the drawings. Devices 200, 400, 500, 600, and 900 and their descriptions), the entire contents of which are incorporated herein by reference.

In some embodiments of the DEP configuration microfluidic device, the upper electrode 210 is part of the first wall (or cover 110) of the enclosure 102, and the electrode activation substrate 206 and the lower electrode 204 are of the enclosure 102. It is part of the second wall (or support structure 104). The region / chamber 202 can be between the first wall and the second wall. In other embodiments, the electrode 210 is part of a second wall (or support structure 104) and one or both of the electrode activating substrate 206 and / or the electrode 210 is the first wall (or cover 110). ) Is part of. Further, the light source 216 can be used as an alternative to illuminate the enclosure 102 from below.

Using the microfluidic device 200 of FIGS. 1B and 1C having a DEP configuration, the prime mover module 162 projects an optical pattern 218 onto the microfluidic device 200 to enclose and capture a small object (eg, a square pattern 220). By activating one or more of the first set of DEP electrodes in the DEP electrode region 214a of the inner surface 208 of the electrode activation substrate 206, microobjects in the medium 180 in the region / chamber 202 (shown). Can be selected. The prime mover module 162 then moves the light pattern 218 relative to the microfluidic device 200 to activate one or more of the second set of DEP electrodes in the DEP electrode region 214. It is possible to move minute objects generated and captured by the chew. Alternatively, the microfluidic device 200 can be moved relative to the light pattern 218.

In another embodiment, the microfluidic device 200 can have a DEP configuration that is independent of photoactivation of the DEP electrode on the inner surface 208 of the electrode activation substrate 206. For example, the electrode activating substrate 206 may include selectively addressable and energizable electrodes located opposite the surface containing at least one electrode (eg, cover 110). A switch (eg, a transistor switch on a semiconductor substrate) can be selectively opened and closed to activate or deactivate the DEP electrode in the DEP electrode region 214, thereby activating or deactivating the region in the vicinity of the activated DEP electrode / Generates a net DEP force on microscopic objects (not shown) in chamber 202. Depending on features such as the frequency of the power supply 212 and the medium (not shown) and / or the dielectric properties of the micro-objects in the region / chamber 202, the DEP force can attract or reject nearby micro-objects. .. Capturing one or more microobjects in region / chamber 202 by selectively activating or deactivating a set of DEP electrodes (eg, in a set of DEP electrode regions 214 forming a square pattern 220). Can be moved within the region / chamber 202. The prime mover module 162 of FIG. 1A controls such a switch, thus activating and deactivating the individual electrodes of the DEP electrode to deactivate certain micro-objects (not shown) around the region / chamber 202. It can be selected, captured, and moved. Microfluidic devices having a DEP configuration that includes selectively addressable and energizable electrodes are known in the art and are described, for example, in US Pat. Nos. 6,294,063 (Becker et al.) And 6. , 942, 776 (Medoro), the entire contents of which are incorporated herein by reference.

As a further alternative, the microfluidic device 200 can have an electronic wetting (EW) configuration, the EW configuration may be an alternative to the DEP configuration, or a microfluidic separate from the portion having the DEP configuration. It may be arranged in a part of the device 200. The EW configuration can be a photoelectron wetting configuration or an electron wetting (EWOD) configuration on a dielectric, both of which are known in the art. In some EW configurations, the support structure 104 has an electrode activating substrate 206 sandwiched between a dielectric layer (not shown) and a lower electrode 204, as described below. The dielectric layer can include hydrophobic materials and / or can be coated with hydrophobic materials. In the case of the microfluidic device 200 having an EW configuration, the inner surface 208 of the support structure 104 is the inner surface of the dielectric layer or its hydrophobic coating.

The dielectric layer (not shown) can include one or more oxide layers and can have a thickness of about 50 nm to about 250 nm (eg, about 125 nm to about 175 nm). In certain embodiments, the dielectric layer can include a layer of oxide such as a metal oxide (eg, aluminum oxide or hafnium oxide). In certain embodiments, the dielectric layer can include dielectric materials other than metal oxides such as silicon oxide or nitrides. Regardless of the exact composition and thickness, the dielectric layer can have an impedance of about 10 k ohms to about 50 k ohms.

In some embodiments, the surface of the dielectric layer facing inward towards the region / chamber 202 is coated with a hydrophobic material. Hydrophobic materials can include, for example, fluorinated carbon molecules. Examples of fluorinated carbon molecules include perfluoropolymers such as polytetrafluoroethylene (eg, TEFLON® or poly (2,3-difluoromethylenel-perfluorotetrahydrofuran)) (eg, CYTOP ™). The molecules constituting the hydrophobic material can be covalently bonded to the surface of the dielectric layer. For example, the molecules of the hydrophobic material are attached to the surface of the dielectric layer by a linker such as a siloxane group, a phosphonic acid group, or a thiol group. Thus, in some embodiments, the hydrophobic material can include alkyl-terminated siloxane, alkyl-terminated phosphonic acid, or alkyl-terminated thiol. The alkyl group is a long-chain hydrocarbon (eg, at least 10). It can be (with at least 16, 18, 20, 22 or more carbon chains) or an alkyl group of fluorinated (or perfluoroylated) carbon chains. Thus, for example, the hydrophobic material can include fluoroalkyl-terminated siloxane, fluoroalkyl-terminated phosphonic acid, or fluoroalkyl-terminated thiol. In some embodiments, the hydrophobic coating. Has a thickness of about 10 nm to about 50 nm. In other embodiments, the hydrophobic coating has a thickness of less than 10 nm (eg, less than 5 nm or about 1.5 to 3.0 nm).

In some embodiments, the cover 110 of the microfluidic device 200 having an electronic wetting configuration is also coated with a hydrophobic material (not shown). The hydrophobic material can be the same hydrophobic material used to coat the dielectric layer of the support structure 104, and the hydrophobic coating is approximately the same as the thickness of the hydrophobic coating on the dielectric layer of the support structure 104. It can have a certain thickness. Further, the cover 110 may include an electrode activating substrate 206 sandwiched between the dielectric layer and the upper electrode 210 in the form of a support structure 104. The dielectric layer of the electrode activating substrate 206 and the cover 110 can have the same composition and / or dimensions as the dielectric layer of the electrode activating substrate 206 and the support structure 104. Therefore, the microfluidic device 200 can have two electron wetting surfaces.

In some embodiments, the electron activating substrate 206 can include a photoconducting material such as the photoconducting material described above. Thus, in certain embodiments, the electrode activated substrate 206 may comprise or consist of a layer of hydrogenated amorphous silicon (a-Si: H). a-Si: H can contain, for example, about 8% to 40% hydrogen (calculated as the total number of hydrogen atoms and the total number of silicon atoms / the total number of hydrogen atoms multiplied by 100). The layer a—Si: H can have a thickness of about 500 nm to about 2.0 μm. Alternatively, the electron activating substrate 206 can include an electrode controlled by a phototransistor switch (eg, a conductive metal electrode), as described above. Microfluidic devices with photoelectron wetting configurations are known in the art and / or can be constructed using electrode activated substrates known in the art. For example, U.S. Pat. No. 6,958,132 (Chiou et al.), The entire content of which is incorporated herein by reference, discloses a photoelectron wetting configuration with a photoconductive material such as a-Si: H. On the other hand, US Patent Application Publication No. 2014/0124370 (Short et al.) Cited above discloses an electrode activating substrate having an electrode controlled by a phototransistor switch.

Therefore, the microfluidic device 200 can have a photoelectron wetting configuration and the light pattern 218 can be used to activate the photoresponsive EW region or photoresponsive EW electrode on the electrode activation substrate 206. .. Such an activated EW region or EW electrode of the electrode activating substrate 206 produces an electron wetting force on the inner surface 208 of the support structure 104 (ie, the inner surface of the overlapping dielectric layers or its hydrophobic coating). be able to. Droplets (eg, aqueous medium) that come into contact with the inner surface 208 of the support structure 104 by altering the light pattern 218 incident on the electron activation substrate 206 (or moving the microfluidic device 200 relative to the light source 216). , Aqueous solutions or aqueous solvents) can move through immiscible fluids (eg, oil media) present within the region / chamber 202.

In another embodiment, the microfluidic device 200 can have an EWOD configuration and the electrode activating substrate 206 provides light-independent, selectively addressable and energizing electrodes for activation. Can include. Therefore, the electrode activation substrate 206 can include such a patterned electron wetting (EW) electrode. The pattern can be, for example, an array of approximately square EW electrodes arranged in a matrix such as that shown in FIG. 2B. Alternatively, the pattern can be an array of approximately hexagonal EW electrodes forming a hexagonal lattice. Regardless of the pattern, the EW electrode can be selectively activated (or deactivated) by an electric switch (for example, a transistor switch on a semiconductor substrate). By selectively activating and deactivating the EW electrode on the electrode activation substrate 206, droplets (not shown) that come into contact with the inner surface 208 of the overlapping dielectric layers or its hydrophobic coating are located in the region / chamber. You can move within 202. The prime mover module 162 of FIG. 1A can control such a switch, thus activating and deactivating individual EW electrodes to select and move specific droplets around the region / chamber 202. be able to. Microfluidic devices having an EWOD configuration with selectively addressable and energizable electrodes are known in the art and are described, for example, in US Pat. No. 8,685,344 (Sundarsan et al.). The entire contents are incorporated herein by reference.

Regardless of the configuration of the microfluidic device 200, the power supply 212 can be used to provide a potential (eg, an AC power supply potential) that feeds the electrical circuit of the microfluidic device 200. The power supply 212 may be the same as the power supply 192 referenced in FIG. 1 or a component of the power supply 192. The power supply 212 may be configured to provide an AC voltage and / or current to the upper electrode 210 and the lower electrode 204. In the case of AC voltage, the power supply 212 captures and moves individual micro-objects (not shown) within the region / chamber 202, as described above, and / or, as also described above, the region / chamber 202. Generates a net DEP force (or electron wetting force) strong enough to alter the wetting properties of the inner surface 208 of the inner support structure 104 (ie, the dielectric layer and / or the hydrophobic coating on the dielectric layer). Sufficient frequency range and average or peak power (eg, voltage or current) can be provided. Such frequency ranges and average or peak power ranges are known in the art. For example, US Pat. No. 6,958,132 (Chiou et al.), US Pat. No. RE44,711 (Wu et al.) (Originally issued as US Pat. No. 7,612,355), and US Patent Application Publication. 2014/0124370 (Short et al.), 2015/036598 (Khandros et al.)
, And 2015/0306599 (Khandros et al.).

Quarantine enclosure. Non-limiting examples of common isolation enclosures 224, 226, and 228 are shown within the microfluidic device 230 shown in FIGS. 2A-2C. Each isolation enclosure 224, 226, and 228 can include a separation structure 232 that defines the separation zone 240 and the contiguous zone 236 that fluidly connects the isolation zone 240 to the channel 122. Contiguous zone 236 can include a proximal opening 234 to the microfluidic channel 122 and a distal opening 238 to the separation zone 240. Contiguous zone 236 may be configured such that the maximum penetration depth of the flow of fluid medium (not shown) flowing into the isolation enclosure 224, 226, 228 from the microfluidic channel 122 does not reach within the separation zone 240. Thus, due to contiguous zone 236, microobjects (not shown) or other materials (not shown) placed within isolation zone 240 of isolation enclosures 224, 226, 228 are medium 180 in channel 122. Can be separated from the flow of and substantially unaffected by the flow of medium 180 in the microfluidic channel 122.

The isolation enclosures 224, 226, and 228 of FIGS. 2A-2C each have a single opening that opens directly to the microfluidic channel 122. The opening of the isolation enclosure opens laterally from the microfluidic channel 122. The electrode activation substrate 206 is under both the microfluidic channel 122 and the isolation enclosures 224, 226, and 228. The top surface of the electrode activation substrate 206 in the enclosure of the isolation enclosure, which forms the floor of the isolation enclosure, forms the floor of the flow channel (or flow region, respectively) of the microfluidic device, where the microfluidic channel 122 (or channel is present). If not, it is arranged at the same height as or substantially the same height as the upper surface of the electrode activation substrate 206 in the flow region). The electrode activated substrate 206 may be featureless or less than about 3 μm, less than about 2.5 μm, less than about 2 μm, less than about 1.5 μm, less than about 1 μm, less than about 0.9 μm, about 0. It may have an irregular or patterned surface that varies from less than 5 μm, less than about 0.4 μm, less than about 0.2 μm, less than about 0.1 μm, or less, from the highest ridge to the lowest depression. Fluctuations in the elevation of the top surface of the substrate across both the microfluidic channel 122 (or flow region) and the isolation enclosure are less than about 3%, less than about 2% of the wall height of the isolation enclosure or the wall height of the microfluidic device. , Less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.5%, less than about 0.3%, or less than about 0.1%. Although the microfluidic device 200 has been described in detail, this also applies to any microfluidic device 100, 230, 250, 280, 290, 320, 400, 450, 500, 700 described herein.

Thus, the microfluidic channel 122 can be an example of a sweep region, and the isolation region 240 of the isolation enclosures 224, 226, 228 can be an example of a non-sweep region. As mentioned, the microfluidic channels 122 and isolation enclosures 224, 226, 228 may be configured to include one or more fluid media 180. In the example shown in FIGS. 2A-2B, the port 222 can be connected to the microfluidic channel 122 to allow the fluid medium 180 to be introduced into or out of the microfluidic device 230. Prior to introducing the fluid medium 180, the microfluidic device can be primed with a gas such as carbon dioxide gas. When the microfluidic device 230 includes the fluid medium 180, the flow 242 of the fluid medium 180 in the microfluidic channel 122 can be selectively generated and stopped. For example, as shown, ports 222 can be located at different locations (eg, at both ends) of the microfluidic channel 122, from one port 222 acting as an inlet to another port 222 acting as an outlet. A medium flow 242 can be produced.

FIG. 2C shows a detailed view of an example of the isolation enclosure 224 according to the present disclosure. An example of a micro object 246 is also shown.

As is known, the flow 242 of the fluid medium 180 in the microfluidic channel 122 beyond the proximal opening 234 of the isolation enclosure 224 results in a secondary flow 244 of the medium 180 in and / or out of the isolation enclosure 224. Can be made to. _{Length L con of the contiguous} zone 236 of the isolation enclosure 224 (ie, from the proximal opening 234 to the tip opening 238) to separate the microobjects 246 in the isolation zone 240 of the isolation enclosure 224 from the secondary flow 244. Should be greater than _{the penetration depth D p} of the secondary flow 244 into the continental zone 236. The depth _{D p} penetration of the secondary flow 244 is associated with the configuration of the speed and the microfluidic channel 122 and a microfluidic channel 122 proximal end opening portion 234 of the connection region 236 to the fluid medium 180 flowing in microfluidic channel 122 It depends on various parameters. In a given microfluidic device, the configuration of the microfluidic channel 122 and the opening 234 is fixed, while the velocity of the flow 242 of the fluid medium 180 within the microfluidic channel 122 is variable. Therefore, for each isolation enclosure 224, the maximum velocity Vmax of the flow 242 of the fluid medium 180 in the channel 122 is identified, which ensures that _{the penetration depth D p} of the secondary flow 244 does not exceed the length L _{con of the contiguous zone 236.} can do. As long as the flow rate of the flow 242 of the fluid medium 180 in the microfluidic channel 122 does not exceed the maximum velocity Vmax, the resulting secondary flow 244 to the microfluidic channel 122 and the contiguous zone 236 can be restricted. It can be prevented from entering the separation region 240. Therefore, the flow 242 of the medium 180 in the microfluidic channel 122 does not draw the microobject 246 out of the separation region 240. Rather, the micro-object 246 disposed within the separation region 240 remains within the separation region 240 regardless of the flow 242 of the fluid medium 180 within the microfluidic channel 122.

Further, as long as the flow rate of the flow 242 of the medium 180 in the microfluidic channel 122 does not exceed Vmax, the flow 242 of the fluid medium 180 in the microfluidic channel 122 may contain various particles (eg, fine particles and / or nanoparticles). Do not move from the microfluidic channel 122 into the isolation area 240 of the isolation enclosure 224. Therefore, by making the length L _con of the connection area 236 greater than the maximum penetration depth D _{p of the} secondary flow 244, one isolation enclosure 224 has a microfluidic channel 122 or another isolation enclosure (eg, FIG. 2D). Contamination by various particles from the isolation enclosure 226,228) can be avoided.

Since the connection region 236 of the microfluidic channel 122 and the isolation enclosure 224, 226, 228 can be influenced by the flow 242 of the medium 180 in the microfluidic channel 122, the microfluidic channel 122 and the connection region 236 are microfluidic. It can be considered as the sweep (or flow) area of the device 230. On the other hand, the isolation area 240 of the isolation enclosure 224, 226, 228 can be considered as a non-sweep (or non-flow) area. For example, the components (not shown) in the first fluid medium 180 in the microfluidic channel 122 substantially pass from the microfluidic channel 122 through the connecting region 236 to the second fluid medium 248 in the separation region 240. Can be mixed with the second fluid medium 248 in the separation region 240 only by diffusion of the components of the first medium 180. Similarly, the components of the second medium 248 (not shown) in the separation region 240 substantially pass from the separation region 240 through the contiguous zone 236 to the first medium 180 in the microfluidic channel 122. Only by diffusion of the components of medium 248 of 2 can it be mixed with the first medium 180 in the microfluidic channel 122. In some embodiments, the degree of fluid medium exchange between the separation region of the isolation enclosure due to diffusion and the flow region is about 90%, about 91%, about 92%, about 93%, about 94 of the fluid exchange. %, About 95%, about 96%, about 97%, about 98%, or higher than about 99%. The first medium 180 may be the same medium as the second medium 248, or may be a different medium. In addition, the first medium 180 and the second medium 248 can start as the same medium and be different (eg, by one or more cells within the isolation region 240 or through the microfluidic channel 122. (Through adjusting the second medium 248 by modifying the flowing medium 180).

Maximum penetration depth D _p of the secondary flow 244 resulting from the flow 242 of the fluid medium 180 in the microfluidic channels 122, as described above, may depend on several parameters. Examples of such parameters are the shape of the microfluidic channel 122 (eg, the channel can direct the medium to the connecting area 236 and divert the medium from the connecting area 236, or to the microfluidic channel 122. The medium can be directed in a direction substantially orthogonal to the proximal opening 234 of the connection region 236), the width _Wch (or cross-sectional area) of the microfluidic channel 122 at the proximal opening 234 and the proximal opening. _{The width W con} (or cross-sectional area) of the connecting region 236 at 234, the velocity V of the flow 242 of the fluid medium 180 in the microfluidic channel 122, the viscosity of the first medium 180 and / or the second medium 248, and the like. Be done.

In some embodiments, the dimensions of the microfluidic channel 122 and the isolation enclosure 224, 226, 228 can be oriented as follows with respect to the vector of the flow 242 of the fluid medium 180 within the microfluidic channel 122: micro The fluid channel width W _ch (or the cross-sectional area of the microfluidic channel 122) can be approximately orthogonal to the flow 242 of the medium 180, and the width W _con (or cross-sectional area) of the connection region 236 at the opening 234 is micro. It can be approximately parallel to the flow 242 of the medium 180 in the fluid channel 122 and / or the length L _con of the connecting region can be approximately orthogonal to the flow 242 of the medium 180 in the microfluidic channel 122. The above is merely an example, and the relative positions of the microfluidic channels 122 and the isolation enclosures 224, 226, 228 may be in other directions with respect to each other.

As shown in FIG. 2C, the width W _con of the connection region 236 can be uniform from the proximal opening 234 to the distal opening 238. _{Thus, the width W con} of the connection region 236 at the tip opening 238 can be within any range identified herein for _{the width W con} of the connection region 236 at the proximal opening 234. _{Alternatively, the width W con} of the connection region 236 at the tip opening 238 can be greater than _{the width W con} of the connection region 236 at the proximal opening 234.

As shown in FIG. 2C, the width of the separation region 240 at the tip opening 238 can be substantially the same as _{the width W con of the proximal region 236 at the proximal opening 234.} Thus, the width of the separation zone 240 at the tip opening 238 can be within any range identified herein for _{the width W con of the connection region 236 at the proximal opening 234.} Alternatively, the width of the separation zone 240 at the tip opening 238 may be greater than or smaller than _{the width W con of the connection region 236 at the proximal opening 234.} Further, the tip opening 238 may be smaller than the proximal opening 234, and the width W _con of the connection region 236 may be narrowed between the proximal opening 234 and the distal opening 238. For example, the contiguous zone 236 can be narrowed between the proximal and distal openings using a variety of different geometries, such as chamfering the contiguous zone and grading the contiguous zone. Further, any portion or sub portion of the connection region 236 may be narrowed (eg, portion of the connection region adjacent to the proximal opening 234).

2D-2F show another exemplary embodiment of the microfluidic device 250 including the microfluidic circuit 262 and the flow channel 264, which are variants of each microfluidic device 100, circuit 132, and channel 134 of FIG. 1A. show. The microfluidic device 250 also has a plurality of isolation enclosures 266, which are additional variants of the isolation enclosures 124, 126, 128, 130, 224, 226, or 228 described above. In particular, the isolation enclosure 266 of the device 250 shown in FIGS. 2D-2F is the device 100, 200, 230, 250, 280, 290, 500, 550, 560, 600, 620, 640, 670, 700, 720, 720, Replaced by any of the isolation enclosures 124, 126, 128, 130, 224, 226, or 228 described above at 750, 760, 780, 808, 810, 812, 900, 1000, 1100, 1200, 1300, 1400, 1500. Please understand that it is possible. Similarly, the microfluidic device 250 is another variant of the microfluidic device 100, the microfluidic devices 100, 200, 230, 250, 280, 290, 500, 550, 560, 600, 620, 640, described above. Same or different DEP configurations as 670, 700, 720, 720, 750, 760, 780, 808, 810, 812, 900, 1000, 1100, 1200, 1300, 1400, 1500 and any other described herein. It can also have microfluidic system components of.

The microfluidic device 250 of FIGS. 2D-2F is a support structure (not visible in FIGS. 2D-2F, but may be the same as or generally similar to the support structure 104 of device 100 shown in FIG. 1A), microfluidics. It comprises a circuit structure 256 and a cover (not visible in FIGS. 2F-2F, but may be the same as or generally similar to the cover 122 of device 100 shown in FIG. 1A). The microfluidic circuit structure 256 includes a frame 252 and a microfluidic circuit material 260, which may be the same as or generally similar to the frame 114 and microfluidic circuit material 116 of device 100 shown in FIG. 1A. As shown in FIG. 2D, the microfluidic circuit 262 defined by the microfluidic circuit material 260 can include multiple channels 264 (two are shown, but there can be more), with channel 264 being A plurality of isolation enclosures 266 are fluidly connected.

Each isolation enclosure 266 can include a separation structure 272, a separation region 270 within the separation structure 272, and a contiguous zone 268. From the proximal opening 274 of the microfluidic channel 264 to the distal opening 276 in the separation structure 272, the connection region 268 fluidly connects the microfluidic channel 264 to the separation zone 270. In general, according to the above considerations of FIGS. 2B and 2C, the flow 278 of the first fluid medium 254 in channel 264 is directed from the microfluidic channel 264 into and / or out of each contiguous zone 268 of the isolation enclosure 266. A secondary flow 282 of medium 254 of 1 can be provided.

As shown in FIG. 2E, the contiguous zone 268 of each isolation enclosure 266 generally includes an area extending between the proximal opening 274 of the channel 264 and the distal opening 276 of the separation structure 272. The length L _con of the contiguous zone 268 can be greater than the maximum penetration depth D _{p of the} secondary flow 282, in which case the secondary flow 282 is not redirected towards the demarcation zone 270 and is in the contiguous zone. Extends within 268 (as shown in FIG. 2D). Alternatively, as shown in FIG. 2F, the connection area 268 _{can have a length L con} that is less than _{the maximum penetration depth D p} , in which case the secondary flow 282 passes through the connection area 268. Extends and redirects towards the demarcation zone 270. In this latter situation, _{the sum of the lengths L c1} and L _c2 of the contiguous zone 268 is greater than the maximum penetration depth D _p , so the secondary flow 282 does not extend into the demarcation zone 270. _{Regardless of whether the length L con} of the connection area 268 is greater than the penetration depth D _{p or whether the sum of the lengths L c1} and L _c2 of the connection area 268 is greater than the penetration depth D _p. Flow 278 of the first medium 254 in channel 264 not exceeding the maximum velocity V _{max results in a secondary flow with penetration depth D p} and micro-objects within the isolation region 270 of isolation enclosure 266 (not shown). However, it may be the same as or generally similar to the micro-object 246 shown in FIG. 2C) is not pulled out of the separation region 270 by the flow 278 of the first medium 254 in channel 264. The flow 278 in the channel 264 also does not draw various materials (not shown) from the channel 264 into the isolation area 270 of the isolation enclosure 266. Therefore, the only mechanism by which the components in the first medium 254 in the microfluidic channel 264 can be moved from the microfluidic channel 264 into the second medium 258 in the isolation region 270 of the enclosure 266 is by diffusion. be. Similarly, diffusion is also the only mechanism by which the components in the second medium 258 in the isolation area 270 of the isolation enclosure 266 can be moved from the separation area 270 into the first medium 254 in the microfluidic channel 264. .. The first medium 254 can be the same medium as the second medium 258, or the first medium 254 can be a different medium than the second medium 258. Alternatively, the first medium 254 and the second medium 258 start from the same medium and change the medium flowing, for example, by one or more cells within the isolation region 270 or through the microfluidic channel 264. Can be different through the preparation of the second medium.

As shown in FIG. 2E, the width W _ch microfluidic channel 264 in the microfluidic channel 264 _(i.e., taken across the direction of fluid medium flow through the microfluidic channel indicated by the arrow 278 in FIG. 2D) _Can be substantially orthogonal to the width W con1 of the proximal opening 274 and therefore can be substantially parallel to _{the width W con 2 of the distal opening 276. However, the width con1 of} the base end opening 274 and the width W _con2 of the tip opening 276 need not be substantially orthogonal to each other. For example, an axis directed width W _con1 the proximal opening 274 (not shown), the angle between the different axes directed width W _con2 the distal opening 276 can be a non-orthogonal, thus , Can be other than 90 °. Examples of alternatively directed angles include angles within any of the following ranges: about 30 ° to about 90 °, about 45 ° to about 90 °, about 60 ° to about 90 °, and the like.

In various embodiments of the isolation enclosure (eg, 124, 126, 128, 130, 224, 226, 228, or 266), the separation region (eg, 240 or 270) is configured to include a plurality of microobjects. NS. In other embodiments, the separation region may be configured to include only one, two, three, four, five, or a similar relatively small number of microobjects. Thus, the volume of the separation region is, for example, at least 1 × 10 ⁶ cubic μm, at least 2 × 10 ⁶ cubic μm, at least 4 × 10 ⁶ cubic μm, at least 6 × 10 ⁶ cubic μm, or greater. obtain.

In various embodiments of the isolation enclosure, the width _{W ch} of proximal end opening portion (e.g., 234) microfluidic channels (e.g., 122) may be in any range of: about 50 to 1000 [mu] m, about 50-500 μm, about 50-400 μm, about 50-300 μm, about 50-250 μm, about 50-200 μm, about 50-150 μm, about 50-100 μm, about 70-500 μm, about 70-400 μm, about 70-300 μm, about 70-250 μm, about 70-200 μm, about 70-150 μm, about 90-400 μm, 90-300 μm, about 90-250 μm, about 90-200 μm, about 90-150 μm, about 100-300 μm, about 100-250 μm, about 100 ~ 200 μm, about 100-150 μm, and about 100-120 μm. In some other embodiments, proximal end opening portion (e.g., 234) microfluidic channels in (e.g., 122) the width _{W ch} of about 200～800Myuemu, about 200～700Myuemu, or a range of about 200~600μm Can be. The above is merely an example, and the width _Wch of the microfluidic channel 122 may be in another range (eg, the range defined by any of the endpoints listed above). Further, the width _Wch of the microfluidic channel 122 can be selected to be in any of these ranges in the region of the microfluidic channel other than the proximal opening of the isolation enclosure.

In some embodiments, the isolation enclosure has a height of about 30-about 200 μm or about 50-about 150 μm. In some embodiments, the isolation enclosure is about 1 × 10 ⁴ to about 3 × 10 ⁶ square μm, about 2 × 10 ⁴ to about 2 × 10 ⁶ square μm, about 4 × 10 ⁴ to about 1 × 10 ⁶ It has a cross-sectional area of about 2 × 10 ⁴ to about 5 × 10 ⁵ square μm, about 2 × 10 ⁴ to about 1 × 10 ⁵ square μm, or about 2 × 10 ⁵ to about 2 × 10 ^{6 square μm.} ..

_{In various embodiments of the isolation enclosure, the height H ch} of the microfluidic channel (eg 122) at the proximal opening (eg 234) can be within any of the following ranges: 20-100 μm, 20- 90 μm, 20-80 μm, 20-70 μm, 20-60 μm, 20-50 μm, 30-100 μm, 30-90 μm, 30-80 μm, 30-70 μm, 30-60 μm, 30-50 μm, 40-100 μm, 40-90 μm, 40-80 μm, 40-70 μm, 40-60 μm, or 40-50 μm. The above is only an example, the height H _ch microfluidic channel (e.g., 122), other range (e.g., range defined by any endpoint that is listed above) may be used. The height H _ch of the microfluidic channel 122 in the region of the microfluidic channel other than the proximal end opening of the isolation enclosure may be selected to be any of these ranges.

In various embodiments of the isolation enclosure, the cross-sectional area of the microfluidic channel (eg, 122) at the proximal opening (eg, 234) can be within any of the following ranges: 500-50,000 square μm, 500 to 40,000 square μm, 500 to 30,000 square μm, 500 to 25,000 square μm, 500 to 20,000 square μm, 500 to 15,000 square μm, 500 to 10,000 square μm, 500 to 7,500 square μm, 500 to 5,000 square μm, 1,000 to 25,000 square μm, 1,000 to 20,000 square μm, 1,000 to 15,000 square μm, 1,000 to 10, 000 square μm, 1,000 to 7,500 square μm, 1,000 to 5,000 square μm, 2,000 to 20,000 square μm, 2,000 to 15,000 square μm, 2,000 to 10, 000 square μm, 2,000 to 7,500 square μm, 2,000 to 6,000 square μm, 3,000 to 20,000 square μm, 3,000 to 15,000 square μm, 3,000 to 10, 000 square μm, 3,000 to 7,500 square μm, or 3,000 to 6,000 square μm. The above is merely an example, and the cross-sectional area of the microfluidic channel (eg, 122) at the proximal opening (eg, 234) is in other ranges (eg, the range defined by any of the endpoints listed above). There may be.

In various embodiments of the isolation enclosure, the length L _con of the contiguous zone (eg, 236) can be within any of the following ranges: about 1-600 μm, 5-550 μm, 10-500 μm, 15-400 μm, 20-300 μm, 20-500 μm, 40-400 μm, 60-300 μm, 80-200 μm, or about 100-150 μm. The above is merely an example, and the length L _con of the continental zone (eg, 236) can be within a range different from that of the above example (eg, the range defined by any of the endpoints listed above).

In various embodiments of the isolation enclosure, proximal end opening portion (e.g., 234) connection region (e.g., 236) the width _{W con} of, may be within any range below: 20~500μm, 20~400μm 20,300 μm, 20 to 200 μm, 20 to 150 μm, 20 to 100 μm, 20 to 80 μm, 20 to 60 μm, 30 to 400 μm, 30 to 300 μm, 30 to 200 μm, 30 to 150 μm, 30 to 100 μm, 30 to 80 μm, 30 -60 μm, 40-300 μm, 40-200 μm, 40-150 μm, 40-100 μm, 40-80 μm, 40-60 μm, 50-250 μm, 50-200 μm, 50-150 μm, 50-100 μm, 50-80 μm, 60-200 μm , 60-100 μm, 60-100 μm, 60-80 μm, 70-150 μm, 70-100 μm, and 80-100 μm. _{The above is merely an example, and the width W con} of the contiguous zone (eg, 236) of the proximal opening (eg, 234) can be different from the example above (eg, defined by any of the endpoints listed above). Range).

_{In various embodiments of the isolation enclosure, the width W con} of the connecting region (eg, 236) at the proximal opening (eg, 234) is a microobject (eg, T cell, B cell, egg) for which the isolation enclosure is intended. , Or a living cell that can be an embryo), which can be at least as large as the maximum size. _{For example, the width W con} of the contiguous zone 236 at the proximal opening 234 of the isolation enclosure in which the oocyte, egg, or embryo is placed can be in any of the following ranges: about 100 μm, about 110 μm, about 120 μm, About 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, about 200 μm, about 225 μm, about 250 μm, about 300 μm, or about 100-400 μm, about 120-350 μm, about 140-200-200 300 μm, or about 140-200 μm. _{The above is merely an example, and the width W con} of the contiguous zone (eg, 236) at the proximal opening (eg, 234) may be different from the example above (eg, defined by any of the endpoints listed above). Range).

In various embodiments of the isolation enclosure, the width _Wpr of the proximal opening of the connection region is at least as large as the maximum dimension of the micro-object for which the isolation enclosure is intended (eg, a biological micro-object such as a cell). Can be. For example, the width W _pr is about 50 μm, about 60 μm, about 100 μm, about 200 μm, about 300 μm, or about 50-300 μm, about 50-200 μm, about 50-100 μm, about 75-150 μm, about 75-100 μm, or about. It can be in the range of 200-300 μm.

In various embodiments of the isolation enclosure, _{the ratio of the length L con of the connection region (eg, 236) to the width W con} of the connection region (eg, 236) at the proximal opening 234 is greater than or equal to any of the following ratios: Can be: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7. A ratio of 0, 8.0, 9.0, 10.0, or more. The above is merely an example, _{and the ratio of the length L con of the connection region 236 to the width W con} of the connection region 236 at the proximal opening 234 may differ from the above example.

Various embodiments of microfluidic devices 100, 200, 230, 250, 280, 290, 500, 550, 560, 600, 620, 640, 670, 700, 720, 720, 750, 760, 780, 808, 810, At 812, 900, 1000, 1100, 1200, 1300, 1400, 1500, V _max is about 0.2 microliter / sec, about 0.3 microliter / sec, about 0.4 microliter / sec, about 0. .5 microliters / second, about 0.6 microliters / second, about 0.7 microliters / second, about 0.8 microliters / second, about 0.9 microliters / second, about 1.0 microliters / second Set to about 1.1 microliters / second, about 1.2 microliters / second, about 1.3 microliters / second, about 1.4 microliters / second, or about 1.5 microliters / second. be able to.

In various embodiments of a microfluidic device having an isolation enclosure, the volume of the isolation enclosure isolation region (eg, 240) is, for example, at least 5 × 10 ⁵ cubic μm, at least 8 × 10 ⁵ cubic μm, and at least 1 × 10 ⁶ cubic μm, at least 2 × 10 ⁶ cubic μm, at least 4 × 10 ⁶ cubic μm, at least 6 × 10 ⁶ cubic μm, at least 8 × 10 ⁶ cubic μm, at least 1 × 10 ⁷ cubic μm, at least 5 × 10 ⁷ cubic The volume can be μm, at least 1 × 10 ⁸ cubic μm, at least 5 × 10 ⁸ cubic μm, at least 8 × 10 ⁸ cubic μm, or more. In various embodiments of a microfluidic device having an isolation enclosure, the volume of the isolation enclosure is about 5 × 10 ⁵ cubic μm, about 6 × 10 ⁵ cubic μm, about 8 × 10 ⁵ cubic μm, about 1 × 10 ⁶ cubic. μm, about 2 × 10 ⁶ cubic μm, about 4 × 10 ⁶ cubic μm, about 8 × 10 ⁶ cubic μm, about 1 × 10 ⁷ cubic μm, about 3 × 10 ⁷ cubic μm, about 5 × 10 ⁷ cubic μm, Alternatively, it can have a volume of about 8 × 10 ^{7 cubic μm or more.} In some other embodiments, the volume of the isolation enclosure is from about 1 nanoliter to about 50 nanoliters, 2 nanoliters to about 25 nanolitres, 2 nanoliters to about 20 nanoliters, about 2 nanoliters to about 15 It can be nanoliters, or about 2 nanoliters to about 10 nanoliters.

In various embodiments, the microfluidic device has an isolation enclosure configured as in any of the embodiments described herein, in which case the microfluidic device is about 5 to about 10. Isolation enclosure, about 10 to about 50 isolation enclosures, about 100 to about 500 isolation enclosures, about 200 to about 1000 isolation enclosures, about 500 to about 1500 isolation enclosures, about 1000 to about 2000 isolation enclosures It has a quarantine enclosure, or about 1000 to about 3500 quarantine enclosures. The isolation enclosures do not have to be all the same size and may include different configurations (eg, different widths, different features within the isolation enclosure).

FIG. 2G shows a microfluidic device 280 according to one embodiment. The microfluidic device 280 is shown in FIG. 2G and is a stylized view of the microfluidic device 100. In practice, the microfluidic device 280 and its components (eg, channels 122 and isolation enclosure 128) have the dimensions discussed herein. The microfluidic circuit 120 shown in FIG. 2G has two ports 107, four separate channels 122, and four separate flow paths 106. The microfluidic device 280 further includes a plurality of isolation enclosures leading to each channel 122. In the microfluidic device shown in FIG. 2G, the isolation enclosure has a geometry similar to that of the enclosure shown in FIG. 2C and therefore has both a contiguous zone and a separation zone. Thus, the microfluidic circuit 120, sweep areas (e.g., the maximum penetration depth _D portion of the connection region 236 in the _p channel 122 and secondary flow 244) and non-sweeping region (e.g., isolation region 240 and the secondary flow 244 including both the maximum penetration depth D _p-free portion of the connection region 236 in) of.

Coating Solution and Coating Agent Maintenance of biological micro-objects (eg, living cells) within a microfluidic device (eg, DEP-constituting and / or EW-constituting microfluidic device), without the intention of being limited by theory, is micro. At least one or more inner surfaces of the fluid device present a layer of organic and / or hydrophilic molecules that provides the main interface between the microfluidic device and the biological microobjects maintained within it. Can be facilitated when conditioned or coated (ie, biological microobjects exhibit increased viability, greater proliferation, and / or greater portability within the microfluidic device). In some embodiments, one or more of the inner surfaces of the microfluidic device (eg, the inner surface of the electrode activation substrate of the DEP-constituting microfluidic device, the microfluidic cover, and / or the surface of the circuit material) are organic molecules. And / or can be treated or modified with a coating solution and / or coating agent to produce the desired layer of hydrophilic molecules.

The coating may be applied before or after the introduction of the biological micro-object, or may be introduced at the same time as the biological micro-object. In some embodiments, the biological microobject can be carried into a fluid medium containing one or more dressings of the microfluidic device. In other embodiments, the inner surface of a microfluidic device (eg, a DEP-constituting microfluidic device) is treated or "primed" with a coating solution containing a coating agent prior to introducing the biological microobject into the microfluidic device. Is done.

In some embodiments, at least one surface of the microfluidic device provides a layer of organic and / or hydrophilic molecules suitable for the maintenance and / or growth of biological microobjects (eg, as described below). Includes a coating (which provides a flexible adjustment surface). In some embodiments, substantially all inner surfaces of the microfluidic device include a coating material. The coated inner surface may include the surface of a flow region (eg, a channel), the surface of a chamber, the surface of an isolation enclosure, or a combination thereof. In some embodiments, each of the isolation enclosures has at least one inner surface coated with a coating material. In other embodiments, each of the plurality of flow regions or channels has at least one inner surface coated with a coating material. In some embodiments, at least one inner surface of each of the plurality of isolation enclosures and each of the plurality of channels is coated with a coating material.

Coatings / Solutions Any conventional coating / coating solution may be used, including but not limited to serum or serum factors, bovine serum albumin (BSA), polymers, detergents, enzymes, and any combination thereof. ..

Polymer-based coating material At least one inner surface may include a coating material containing a polymer. The polymer may be covalently or non-covalently attached (or non-specifically attached) to at least one surface. Polymers can have a wide variety of structural motifs, such as those found in block polymers (and copolymers), star polymers (star copolymers), and graft or comb polymers (graft copolymers), all of which are described herein. It may be suitable for the disclosed method.

The polymer may include a polymer containing an alkylene ether moiety. A wide range of alkylene ether-containing polymers may be suitable for use in the microfluidic devices described herein. A non-limiting and exemplary class of alkylene ether-containing polymers includes blocks of polyethylene oxide (PEO) and polypropylene oxide (PPO) subunits in different proportions and locations within the polymer chain. It is an ion block copolymer. Pluronic® Polymer (BASF) is a block copolymer of this type and is known in the art to be suitable for use in contact with living cells. Polymer has an average molecular weight _{M W,} can range from about 2000Da~ about 20 KDa. In some embodiments, the PEO-PPO block copolymer can have a hydrophilic lipophilic balance (HLB) greater than about 10 (eg, 12-18). Specific Pluronic® polymers useful to provide a coated surface include Pluronic® L44, L64, P85, and F127 (including F127NF). Alkylene ether containing polymer of another class is a polyethylene glycol _(PEG M W <100,000Da) or alternatively polyethylene oxide _{(PEO, M W> 100,000)} . In some embodiments, PEG can have about 1000 Da, 5000 Da, 10,000 Da, or 20,000Da of _{M W.}

In other embodiments, the coating material may include a polymer containing a carboxylic acid moiety. The carboxylic acid subunit can be an alkyl, alkenyl, or aromatic portion-containing subunit. A non-limiting example is polylactic acid (PLA). In other embodiments, the coating material may comprise a polymer comprising a phosphate moiety to either the pen setup from the polymer backbone terminated or polymer backbone. In yet another embodiment, the coating material may include a polymer containing a sulfonic acid moiety. The sulfonic acid subunit can be an alkyl, alkenyl, or aromatic portion-containing subunit. A non-limiting example is polystyrene sulfonic acid (PSSA) or polyanethole sulfonic acid. In a further embodiment, the coating material may include a polymer containing an amine moiety. Polyaminopolymers can include natural polyamine polymers or synthetic polyamine polymers. Examples of natural polyamines include spermine, spermidine, and putrescine.

In other embodiments, the coating material may include a polymer containing a saccharide moiety. In a non-limiting example, polysaccharides such as xanthan gum or dextran may be suitable for forming materials that can reduce or prevent cell puncture in microfluidic devices. For example, a dextran polymer having a size of about 3 kDa may be used to provide a coating material for the surface within a microfluidic device.

In other embodiments, the coating material may comprise a nucleotide moiety, i.e. a polymer containing nucleic acid, which has a ribonucleotide moiety or a deoxyribonucleotide moiety and may provide a polyelectrolyte surface. Nucleic acid may comprise only natural nucleotide moiety, or without limitation, 7-deazaadenine, pen toast, methylphosphonic acid, or a nucleic acid base such as phosphorothioate moieties, ribose, non-natural nucleotide moiety comprising a phosphate moiety analogs It may be included.

In yet another embodiment, the coating material may include a polymer containing an amino acid moiety. Polymers containing amino acid moieties may include natural amino acid-containing polymers or unnatural amino acid-containing polymers, any of which may include peptides, polypeptides, or proteins. In a non-limiting example, as a dressing, the protein is bovine serum albumin (BSA) and / or serum containing albumin and / or one or more other similar proteins (or a combination of several different sera). Can be. Serum can be from any convenient source, including but not limited to fetal bovine serum, sheep serum, goat serum, horse serum and the like. In certain embodiments, the BSA in the coating solution is 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg. It is present in the range of about 1 mg / mL to about 100 mg / mL, including / mL, or any value above or between them. In certain embodiments, the serum in the coating solution is about 20% (v), including 25%, 30%, 35%, 40%, 45%, or more, or any value in between. / V) can be present in the range of about 50% v / v. In some embodiments, BSA may be present in the coating solution at 5 mg / mL, while in other embodiments BSA may be present in the coating solution at 70 mg / mL. .. In certain embodiments, the serum is present as a dressing in the coating solution at 30%. In some embodiments, extracellular matrix (ECM) proteins may be provided within the coating material to optimize cell attachment to Foster cell growth. Cellular matrix proteins that can be included in the coating material include, but are not limited to, collagen, elastin, RGD-containing peptides (eg, fibronectin), or laminin. In yet other embodiments, growth factors, cytokines, hormones, or other cell signaling species may be provided within the coating material of the microfluidic device.

In some embodiments, the coating material may comprise a polymer comprising two or more of an alkylene oxide moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphate moiety, a saccharide moiety, a nucleotide moiety, or an amino acid moiety. In other embodiments, the conditioned surface of the polymer can be incorporated into the coating material independently or simultaneously, with an alkylene oxide moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphate moiety, a saccharide moiety, a nucleotide moiety, and /. Alternatively, it may contain a mixture of two or more polymers each having an amino acid moiety.

Covalently Bonded Coating Material In some embodiments, at least one inner surface provides a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological microobjects within the microfluidic device. , Containing covalent molecules that provide a regulatory surface for such cells.

The covalently bonded molecule comprises a binding group, which is covalently attached to one or more surfaces of the microfluidic device, as described below. The linking group is also covalently attached to a moiety that is configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / proliferation of biological microobjects.

In some embodiments, covalent moieties configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological microobjects are alkyl or fluoroalkyl (perfluoroalkyl). (Including) portion; mono or polysaccharide (including, but not limited to, dextran); alcohol (including, but not limited to, propargyl alcohol); polyalcohol including, but not limited to, polyvinyl alcohol; polyethylene glycol. , But not limited to alkylene ethers; high molecular weight electrolytes (including, but not limited to, polyacrylic acid or polyvinylphosphonic acid); amino groups (alkylated amines, morphornyls, piperazinyl, etc., but not limited to these). Hydroxyalkylated amino groups, guanidinium groups, heterocyclic groups, etc. containing, but not limited to non-aromaticized nitrogen ring atoms, but not limited to derivatives thereof); phosphoric acid (carboxylate anionic surface). Carboxylic acids including, but not limited to; phosphonic acids (which may provide a phosphonate anion surface); phosphonic acids; sulfonate anions; carboxybetaines; sulfobetaines; Sulfamic acid; or may contain amino acids.

In various embodiments, covalent moieties configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / proliferation of biological microobjects in microfluidic devices are alkyl moieties, fluoroalkyls. Substituted alkyl moieties such as moieties (including, but not limited to, perfluoroalkyl moieties), amino acid moieties, alcohol moieties, amino moieties, carboxylic acid moieties, phosphonic acid moieties, sulfonic acid moieties, sulfamic acid moieties, saccharide moieties, etc. May include non-polymeric moieties. Alternatively, the covalent moiety may include a polymeric moiety that can be any of the aforementioned moieties.

In some embodiments, the covalent moiety forms a linear chain (eg, a linear chain of at least 10 carbons or at least 14, 16, 18, 20, 22, or more carbons). It can contain carbon atoms and can be a linear alkyl moiety. In some embodiments, the alkyl group may comprise a substituted alkyl group (eg, some of the carbons of the alkyl group can be fluorinated or perfluorolated). In some embodiments, the alkyl group may include a first segment that may contain a perfluoroalkyl group that is attached to a second segment that may contain an unsubstituted alkyl group. The first and second segments can be bonded directly or indirectly (eg, by an ether bond), where the first segment of the alkyl group can be located at the tip of the bonding group and of the alkyl group. The second segment may be located at the proximal end of the binding group.

In other embodiments, the covalent moiety may comprise at least one amino acid, which may include two or more types of amino acids. Therefore, the covalent moiety may comprise a peptide or protein. In some embodiments, the covalent moiety may contain amino acids that can provide a zwitterionic surface and support cell growth, survival, portability, or any combination thereof.

In other embodiments, the covalent moiety may comprise at least one alkylene oxide moiety and may include any of the alkylene oxide polymers described above. A useful class of alkylene ether-containing polymers is polyethylene glycol (PEG M _w <100,000 Da) or, alternative, polyethylene oxide (PEO, M _w > 100,000). In some embodiments, the PEG can have _{M w} of about 1000 Da, about 5000 Da, about 10,000 Da, or about 20,000 Da.

The covalent moiety may contain one or more saccharides. The covalent saccharide can be mono, di, or polysaccharide. Covalent saccharides can be modified to introduce reaction pair moieties that allow binding or processing to adhere to the surface. An exemplary reaction pair moiety may include an aldehyde, alkyne, or halo moiety. Polysaccharides can be modified randomly, each saccharide monomer may be modified, or only a portion of the saccharide monomer within the polypeptide can provide a reaction pair moiety that can be attached directly or indirectly to the surface. Is qualified to. One example may include a dextran polysaccharide that can be indirectly attached to the surface via a linear linker.

The covalent moiety may contain one or more amino groups. The amino group can be a substituted amine moiety, a guanidine moiety, a nitrogen-containing heterocyclic moiety or a heteroaryl moiety. The amino-containing moiety may have a structure that allows pH changes in the environment within the microfluidic device and optionally within the isolation enclosure and / or flow region (eg, channel).

The coating material that provides the conditioning surface may include only one type of covalent moiety, or may include two or more different types of covalent moieties. For example, fluoroalkyl-adjusted surfaces (including perfluoroalkyl) are all the same, eg, multiple covalent bonds with the same bond groups and covalent bonds to the surface, the same overall length, and the same number of fluoromethylene units containing fluoroalkyl moieties. Can have parts. Alternatively, the coating material may have two or more covalent moieties that adhere to the surface. For example, the coating material may contain molecules with covalent alkyl or fluoroalkyl moieties having a specified number of methylene or fluoromethylene units, which may provide the ability to present a more bulky moiety on the coated surface. It may further comprise an additional set of molecules with charged moieties covalently attached to an alkyl or fluoroalkyl chain having more methylene or fluoromethylene units. In this case, the first set of molecules, which have different, sterically less demanding ends and fewer skeletal atoms, functionalize the entire surface of the substrate, thereby forming the silicon / silicon oxide, oxidation of the substrate itself. It can help avoid unwanted adhesion or contact with hafnium, or alumina. In another example, the covalent moiety may provide a zwitterionic surface that randomly presents AC charges on the surface.

Adjusted surface properties Other than the adjusted surface composition, other factors such as the physical thickness of the hydrophobic material can affect the DEP force. Various factors such as the mode in which the adjusted surface is formed on the substrate (eg, vapor deposition, liquid phase deposition, spin coating, flooding, and electrostatic coating) can change the physical thickness of the adjusted surface. In some embodiments, the conditioning surface has a thickness of about 1 nm to about 10 nm, about 1 nm to about 7 nm, about 1 nm to about 5 nm, or any individual value between them. In other embodiments, the adjustment surface formed by the covalent moieties can have a thickness of about 10 nm to about 50 nm. In various embodiments, the adjustment surfaces prepared as described herein have a thickness of less than 10 nm. In some embodiments, the covalent portion of the conditioning surface can form a monolayer when covalently bonded to the surface of a microfluidic device (eg, the surface of a DEP constituent substrate) and is less than 10 nm (eg, less than 5 nm or about. It can have a thickness of 1.5 nm to 3.0 nm). These values are in contrast to the thickness of CYTOP® (Asahi Glass Co., Ltd., Japan) fluoropolymer spin coatings, which have thicknesses in the range of about 30 nm. In some embodiments, the conditioning surface does not require a fully formed monolayer in order to be reasonably functional for operation within the DEP-configured microfluidic device.

In various embodiments, the coating material that provides the conditioning surface of the microfluidic device may provide the desired electrical properties. Unintentionally limited by theory, one factor that affects the toughness of a surface coated with a particular coating material is essentially charge capture. Different coating materials can capture electrons, which can lead to destruction of the coating material. Defects in the coating material can increase charge capture and lead to further destruction of the coating material. Similarly, different coating materials have different dielectric strengths (ie, the minimum applied electric field that causes dielectric breakdown), which can affect charge capture. In certain embodiments, the coating material can have an overall structure (eg, a high density monolayer structure) that reduces or limits its charge capture.

In addition to its electrical properties, the conditioned surface can also have properties that are advantageous for use with biomolecules. For example, a conditioned surface containing a fluorinated (or perfluorolated) carbon chain may provide an advantage over an alkyl-terminated chain in reducing the amount of surface fouling. Surface fouling, as used herein, is indiscriminate on the surface of microfluidic devices, which may include permanent or semi-permanent deposition of biomaterials such as proteins and their waste products, nucleic acids and each waste product. Refers to the amount of material deposited.

Single or multi-part covalent coating material to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological microobjects in microfluidic devices, as described below. It can be formed by the reaction of molecules that already contain the constituents. Alternatively, the covalent coating material itself shares a portion with the surface that is configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological microobjects. It can be formed in a two-part sequence by binding to a bound surface-modifying ligand.

How to Prepare a Covalently Bonded Coating Material In some embodiments, the covalently bonded coating material to the surface of a microfluidic device (including, for example, at least one surface of an isolation enclosure and / or flow region) is of formula 1. Or it has the structure of formula 2. The coating material has the structure of Equation 1 when introduced onto the surface in one step, while the coating material has the structure of Equation 2 when introduced in a multi-step processor.

The coating material can be fed-bonded to oxides on the surface of DEP or EW constituent substrates. The DEP or EW constituent substrate may contain silicon, silicon oxide, alumina, or hafnium oxide. The oxide may be present as part of the original chemical structure of the substrate or can be introduced as described below.

The coating material can be attached to the oxide via a bonding group (“LG”), which can be a syloxy or phosphonate ester group formed from the reaction of a siloxane group or phosphonic acid group with the oxide. The portion configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / proliferation of biological microobjects in a microfluidic device can be any portion described herein. .. The linking group LG may be directly or indirectly linked to a moiety configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological microobjects in a microfluidic device. If the binding group LG is directly attached to that moiety, there is no optional linker L and n is 0. If the linking group LG is indirectly connected to the moiety, the linker L is present and n is 1. Linker L is selected from any combination of silicon atom, carbon atom, nitrogen atom, oxygen atom, sulfur atom, and phosphorus atom whose linear skeleton is subject to chemical bond constraints known in the art. It may have a linear portion that may contain 200 non-hydrogen atoms. It is interrupted by any combination of one or more moieties selected from the group consisting of ether groups, amino groups, carbonyl groups, amide groups, or phosphate groups, arylene groups, heteroarylene groups, or heterocyclic groups. obtain. In some embodiments, the backbone of the linker L may contain 10 to 20 carbons. In other embodiments, the backbone of the linker L may comprise from about 5 to about 200 atoms, from about 10 to about 80 atoms, from about 10 to about 50 atoms, or from about 10 to about 40 atoms. In some embodiments, the skeletal atoms are all carbon atoms.

In some embodiments, a portion configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological microobjects is added to the surface of the substrate in a multi-step process. Obtained, it has the structure of formula 2 shown above. This portion can be any portion described above.

In some embodiments, the linking group CG is reacted portion of R _x and paired with reactive moiety R _px result the generated group reaction with _(i.e., reaction portion configured moiety to react with the R _x) Represents. For example, one typical binding group CG may include a carboxamidyl group, which is the result of the reaction of the amino group with a derivative of a carboxylic acid such as an activated ester, acid chloride. Other CGs are formed upon reaction with each reaction moiety paired with a triazolylene group, carboxamidyl, thioamidyl, oxime, mercaptyl, disulfide, ether, or alkenyl group. It may contain any other suitable group, the binding group CG is an organic molecule and / or hydrophilic suitable for the maintenance / growth of biological microobjects in the second end of Linker L (ie, microfluidic device). It can be placed at the end) close to the portion configured to provide a layer of sex molecules, which can include any combination of the elements described above. In some other embodiments, the binding group CG can disrupt the backbone of Linker L. If the binding group CG is triazolylene, it can be the product of a click binding reaction and can be further substituted (eg, a dibenzocyclooctyl melted triazolylene group).

In some embodiments, the coating material (or surface-modifying ligand) is deposited on the inner surface of the microfluidic device using chemical vapor deposition. The vapor deposition process is optionally, for example, by exposure to a solvent bath, sonication, or a combination thereof, a cover 110, a microfluidic circuit material 116, and / or a substrate (eg, an electrode activating substrate of a DEP constituent substrate). It can be improved by cleaning the inner surface 208 of 206 or the dielectric layer of the support structure 104 of the EW constituent substrate in advance. Alternatively or additionally, such pre-cleaning can include treating the cover 110, the microfluidic circuit material 116, and / or the substrate in an oxygen plasma cleaner, which removes various impurities. At the same time, an oxidized surface (eg, an oxide on the surface, which can be covalently modified as described herein) can be introduced. Alternatively, a mixture of hydrochloric acid and hydrogen peroxide or a mixture of sulfuric acid and hydrogen peroxide (eg, a piranha solution in which the ratio of sulfuric acid to hydrogen peroxide can be about 3: 1 to about 7: 1), etc. Liquid phase treatment can also be used in place of the hydrogen peroxide cleaner.

In some embodiments, the microfluidic device 200 may be assembled to form an enclosure 102 defining the microfluidic circuit 120, and then vapor deposition may be used to coat the inner surface of the microfluidic device 200. Unintentionally limiting by theory, depositing such a coating material on a fully assembled microfluidic circuit 120 involves the dielectric layer and / or cover 110 of the microfluidic circuit material 116 and the electrode activating substrate 206. It may be advantageous in avoiding the peeling caused by the weakening of the binding. In embodiments where a two-step process is utilized, the surface modification ligand can be introduced via vapor deposition as described above, followed by organic molecules and / or hydrophilicity suitable for the maintenance / growth of biological microobjects. A portion is introduced that is configured to provide a layer of molecules. Subsequent reactions can be performed by exposing the surface-modified microfluidic device to a suitable binding reagent in solution.

FIG. 2H shows a cross-sectional view of a microfluidic device 290 with an exemplary covalent coating material that provides a conditioning surface. As shown, the coating material 298 (schematically shown) is a high density molecule covalently bonded to both the inner surface 294 of the substrate 286, which can be a DEP substrate, and the inner surface 292 of the cover 288 of the microfluidic device 290. Can include a single layer of. The coating material 298, in some embodiments, comprises the surface of a microfluidic circuit material (not shown) used to define circuit elements and / or structures within the microfluidic device 290, as described above. It can be placed on substantially all inner surfaces 294, 292 that are close to the enclosure 284 of the fluid device 290 and face inward towards the enclosure 284. In an alternative embodiment, the coating material 298 can be placed on only one or several of the inner surfaces of the microfluidic device 290.

In the embodiment shown in FIG. 2H, the coating material 298 can include a single layer of organosiloxane molecules, each molecule covalently attached to the inner surface 292, 294 of the microfluidic device 290 via a siroxylinker 296. Any of the above coating materials 298 can be used (eg, an alkyl-terminated, fluoroalkyl-terminated portion, PEG-terminated portion, dextran-terminated portion, or terminal portion containing a positive or negative charge in the organosiloxane moiety) and terminal. The portion is located at the end facing the enclosure (ie, a single layer portion of the coating material 298 that is not coupled to the inner surfaces 292 and 294 and is close to the enclosure 284).

In other embodiments, the coating material 298 used to coat the inner surface 292, 294 of the microfluidic device 290 can include anionic moieties, cationic moieties, zwitterionic moieties, or any combination thereof. By presenting the cationic, anionic, and / or amphoteric ion moieties on the inner surface of the enclosure 284 of the microfluidic circuit 120, unintentionally limited by theory, the coating material 298 is made of the resulting hydration. With strong water such that water acts as a layer (or "shield") that separates biological micro-objects from interaction with non-biological molecules (eg, silicon and / or silicon oxide in the substrate). Hydrogen bond can be formed. In addition, in embodiments where the coating material 298 is used in combination with a coating agent, the anions, cations, or amphoteric ions of the coating material 298 are non-covalent coatings present in the medium 180 (eg, coating solution) within the enclosure 284. It can form ionic bonds with charged moieties of agents (eg, proteins in solution).

In yet other embodiments, the coating material may contain a hydrophilic coating at the end facing the enclosure or may be chemically modified to present. In some embodiments, the coating material may include an alkylene ether-containing polymer such as PEG. In some embodiments, the coating material may include a polysaccharide such as dextran. Like the charged moieties described above (eg, anionic moiety, cationic moiety, and amphoteric ion moiety), hydrophilic coatings are such that the resulting hydrated water is a non-biological molecule of biological micro-objects. Strong hydrogen bonds with water can be formed that act as a layer (or "shield") that separates from interactions with (eg, silicon and / or silicon oxide in the substrate). Further details of proper coating and modification can be found in U.S. Patent Application Publication No. 15 / 135,707 filed April 22, 2016, which patent application is hereby incorporated by reference in its entirety. It is used for.

Additional System Components to Maintain Cell Viability within Microfluidic Device Isolation Enclosures To promote cell population growth and / or proliferation, additional system components promote the maintenance of functional cells. Can provide environmental conditions. For example, such additional components can provide nutrients, cell growth signaling species, pH regulation, gas exchange, temperature control, and removal of waste products from cells.

System operation and optical control FIGS. 3A-3B are microfluidic devices according to the present disclosure (eg, 100, 200, 230, 250, 280, 290, 500, 550, 560, 600, 620, 640, 670, 700, 720. , 720, 750, 760, 780, 808, 810, 812, 900, 1000, 1100, 1200, 1300, 1400, 1500) and various embodiments of system 150 that can be used for observation. Is shown. As shown in FIG. 3A, the system 150 is a structure (“nested”) configured to hold the microfluidic device 100 (not shown) or any other microfluidic device described herein. ) 300 can be included. The nest 300 can interface with the microfluidic device 320 (eg, an optically actuated electrokinetic device 100) and can provide an electrical connection from the power supply 192 to the microfluidic device 320. 302 can be included. The nest 300 may further include an integrated electrical signal generation subsystem 304. The electrical signal generation subsystem 304 supplies a bias voltage to the socket 302 so that when the microfluidic device 320 is held by the socket 302, a bias voltage is applied across a pair of electrodes in the microfluidic device 320. Can be configured in. Therefore, the electrical signal generation subsystem 304 can be part of the power supply 192. The ability to apply a bias voltage to the microfluidic device 320 does not mean that a bias voltage is applied whenever the microfluidic device 320 is held by the socket 302. Rather, in most cases, the bias voltage is applied intermittently only when necessary to facilitate the generation of dynamic power, such as electrophoresis or electron wetting within the microfluidic device 320.

As shown in FIG. 3A, the nest 300 can include a printed circuit board assembly (PCBA) 322. The electrical signal generation subsystem 304 is mounted on the PCBA 322 and can be electrically integrated in the PCBA 322. An exemplary support also includes a socket 302 mounted on PCBA 322 as well.

Typically, the electrical signal generation subsystem 304 includes a waveform generator (not shown). The electrical signal generation subsystem 304 may further include an oscilloscope (not shown) and / or a waveform amplification circuit (not shown) configured to amplify the waveform received from the waveform generator. The oscilloscope, if present, may be configured to measure the waveform supplied to the microfluidic device 320 held by the socket 302. In certain embodiments, the oscilloscope measures the waveform at the proximal position of the microfluidic device 320 (and at the tip of the waveform generator), thereby being larger in measuring the waveform actually applied to the device. Guarantee accuracy. The data obtained from the oscilloscope measurements may be provided to the waveform generator as feedback, for example, and the waveform generator may be configured to adjust the output based on such feedback. An example of a suitable combined waveform generator and oscilloscope is Red Pitaya ™.

In certain embodiments, the nest 300 further includes a controller 308, such as a microprocessor, used to detect and / or control the electrical signal generation subsystem 304. Examples of suitable microprocessors include Arduino ™ microprocessors such as the Arduino Nano ™. The controller 308 may be used to perform function and analysis, or it may communicate with an external master controller 154 (shown in FIG. 1A) to perform function and analysis. In the embodiment shown in FIG. 3A, the controller 308 communicates with the master controller 154 through an interface 310 (eg, a plug or connector).

In some embodiments, the nest 300 amplifies the waveform generated by the electrical signal generation subsystem 304, including a Red Pitaya ™ waveform generator / oscilloscope unit (“Red Pitaya unit”), and the Red Pitaya unit. , A waveform amplifier circuit that passes the amplification voltage to the microfluidic device 100 can be included. In some embodiments, the Red Pitaya unit measures the amplified voltage at the microfluidic device 320 and then itself as needed so that the measured voltage at the microfluidic device 320 is at the desired value. It is configured to adjust the output voltage of. In some embodiments, the waveform amplifier circuit can have a + 6.5V to -6.5V power supply generated by a pair of DC-DC converters mounted on the PCBA 322, resulting in the microfluidic device 100. Signals up to 13 Vpp are generated.

As shown in FIG. 3A, the support structure 300 (eg, nesting) can further include a thermal control subsystem 306. The thermal control subsystem 306 may be configured to regulate the temperature of the microfluidic device 320 held by the support structure 300. For example, the thermal control subsystem 306 can include a Perche thermoelectric device (not shown) and a cooling unit (not shown). The Perche thermoelectric device can have a first surface configured to interface with at least one surface of the microfluidic device 320. The cooling unit can be, for example, a cooling block (not shown) such as a liquid-cooled aluminum block. The second surface of the Pelche thermoelectric device (eg, the surface opposite to the first surface) may be configured to interface with the surface of such a cooling block. The cooling block can be connected to a fluid path 314 configured to circulate the cooling fluid through the cooling block. In the embodiment shown in FIG. 3A, the support structure 300 receives a cooled fluid from an external reservoir (not shown), including an inlet 316 and an outlet 318, and introduces the cooled fluid into the fluid path 314. Then pass through the cooling block and then return the cooled fluid to the external reservoir. In some embodiments, the Perche thermoelectric device, cooling unit, and / or fluid path 314 can be mounted in case 312 of support structure 300. In some embodiments, the thermal control subsystem 306 is configured to regulate the temperature of the Perche thermoelectric device to achieve the target temperature of the microfluidic device 320. Temperature control of the Pelche thermoelectric device can be achieved, for example, with a thermoelectric power source such as the Pololu ™ thermoelectric power source (Pololu Robotics and Electronics Corp.). The thermal control subsystem 306 can include a feedback circuit such as a temperature value provided by an analog circuit. Alternatively, the feedback circuit may be provided by a digital circuit.

In some embodiments, the nest 300 is a resistor (eg, resistance 1 k ohm +/- 0.1%, temperature coefficient +/- 0.02 ppm / C0) and an NTC thermistor (eg, nominal resistance 1 k ohm +/-). A thermal control subsystem 306 with a feedback circuit that is an analog voltage divider circuit (not shown) including (having 0.01%) can be included. In some cases, the thermal control subsystem 306 measures the voltage from the feedback circuit and then uses the calculated temperature value as an input to the onboard PID control loop algorithm. The output from the PID control loop algorithm can, for example, drive both directional and pulse width modulated signal pins on a Pololu ™ motor drive device (not shown) to activate a thermoelectric power supply. , Control the Perche thermoelectric device.

The nest 300 can include a serial port 350, which allows the microprocessor of controller 308 to communicate with the external master controller 154 via interface 310 (not shown). In addition, the microprocessor of controller 308 can communicate with the electrical signal generation subsystem 304 and the thermal control subsystem 306 (eg, via the Plink tool (not shown)). Therefore, the electrical signal generation subsystem 304 and the thermal control subsystem 306 can communicate with the external master controller 154 via the combination of the controller 308, the interface 310, and the serial port 324. In this way, the master controller 154 can assist the electrical signal generation subsystem 304, in particular by performing scaling calculations for output voltage regulation. A graphical user interface (GUI) (not shown) provided via a display device 170 coupled to an external master controller 154 provides temperature data and waveforms obtained from a thermal control subsystem 306 and an electrical signal generation subsystem 304, respectively. It can be configured to plot the data. Alternatively or additionally, the GUI can allow updates to the controller 308, thermal control subsystem 306, and electrical signal generation subsystem 304.

As mentioned above, the system 150 can include an imaging device 194. In some embodiments, the imaging device 194 includes an optical modulation subsystem 330 (see FIG. 3B). The light modulation subsystem 330 can include a Digital Mirror Device (DMD) or a Microshutter Array System (MSA), either of which receives light from a light source 332 and a subset of the light received by the optics of the microscope 350. It can be configured to send in columns. Alternatively, the optical modulation subsystem 330 is itself such as an organic emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon device (FLCOS), or a transmissive liquid crystal display (LCD). A device that produces light (and thus eliminates the need for a light source 332) can be included. The light modulation subsystem 330 can be, for example, a projector. Therefore, the photomodulation subsystem 330 may be capable of emitting both structured and unstructured light. An example of a suitable optical modulation subsystem 422 is the Mosaic ™ system of Andor Technologies ™. In certain embodiments, the imaging module 164 and / or the prime mover module 162 of the system 150 can control the light modulation subsystem 330.

In certain embodiments, the imaging device 194 further includes a microscope 350. In such an embodiment, the nest 300 and the optical modulation subsystem 330 may be individually configured to be mounted on the microscope 350. The microscope 350 may be configured to be, for example, a standard study grade optical or fluorescence microscope. Thus, the nest 300 may be configured to mount on stage 344 of the microscope 350, and / or the light modulation subsystem 330 may be configured to mount on the port of the microscope 350. In other embodiments, the nesting 300 and the optical modulation subsystem 330 described herein can be integral components of the microscope 350.

In certain embodiments, the microscope 350 may further include one or more detectors 348. In some embodiments, the detector 348 is controlled by the imaging module 164. The detector 348 can include an eyepiece, a charge-coupled device (CCD), a camera (eg, a digital camera), or any combination thereof. If at least two detectors 348 are present, one detector can be, for example, a high frame rate camera, while the other detector can be a sensitive camera. Further, the microscope 350 receives the light reflected and / or emitted from the microfluidic device 320 so that at least a portion of the reflected and / or synchrotron radiation is imaged on one or more detectors 348. It can include configured optical columns. The optical column of the microscope can also include different tube lenses (not shown) for different detectors so that the final magnification at each detector can be different.

In certain embodiments, the imaging device 194 is configured to use at least two light sources. For example, the first light source 332 can be used to generate structural light (eg, via a light modulation subsystem 330) and the second light source 334 can be used to provide unstructured light. The first light source 332 can generate structural light of optically working electrokinesis and / or fluorescence excitation, and the second light source 334 can be used to provide brightfield illumination. In these embodiments, the prime mover module 164 can be used to control the first light source 332 and the imaging module 164 can be used to control the second light source 334. The optical train of the microscope 350 (1) receives the structural light from the optical modulation subsystem 330 and, when the device is held by the nest 300, the structural light at least in a microfluidic device such as an optically actuated electrokinesis device. Concentrate in region 1 and (2) receive reflected and / or radiated light from the microfluidic device and configure it to focus at least a portion of such reflected and / or radiated light on the detector 348. Can be done. The optics can be further configured to receive unstructured light from a second light source and focus the unstructured light to at least a second region of the microfluidic device when the device is held by the nest 300. .. In certain embodiments, the first and second regions of the microfluidic device can be overlapping regions. For example, the first region can be a subset of the second region. In other embodiments, the second light source 334 may additionally or as an alternative include a laser, which may have light of any suitable wavelength. The representation of the optical system shown in FIG. 3B is only a schematic representation, and the optical system may include additional filters, notch filters, lenses, and the like. If the second light source 334 includes one or more light sources for brightfield and / or fluorescence excitation and laser illumination, the physical arrangement of the light sources may differ from the arrangement shown in FIG. 3B and the laser illumination is optical. It can be deployed at any suitable physical location in the system. The approximate positions of the light source 432 and the light source 402 / light modulation subsystem 404 can be exchanged as well.

In FIG. 3B, the first light source 332 is shown to supply light to an optical modulation subsystem 330, which is an optical train of structural light in a microscope 350 of system 355 (not shown). To provide. The second light source 334 is shown to provide unstructured light to the optics via a beam splitter 336. Structural light from the light modulation subsystem 330 and unstructured light from the second light source 334 travel from the beam splitter 336 through the optical train and are provided by the second beam splitter (or light modulation subsystem 330). Depending on the light, it reaches the dichroic filter 338) together, and at the second beam splitter, the light is reflected downwards through the objective lens 336 toward the sample plane 342. Next, the light reflected and / or emitted from the sample plane 342 passes through the objective lens 340 again, passes through the beam splitter and / or the dichroic filter 338, and reaches the dichroic filter 346. Only a part of the light that has reached the dichroic filter 346 is transmitted and reaches the detector 348.

In some embodiments, the second light source 334 emits blue light. With a suitable dichroic filter 346, the blue light reflected from the sample plane 342 can pass through the dichroic filter 346 and reach the detector 348. Conversely, structural light coming from the photomodulation subsystem 330 is reflected from the sample plane 342 but does not pass through the dichroic filter 346. In this example, the dichroic filter 346 filters and removes visible light having wavelengths longer than 495 nm. Such filtering of light from the light modulation subsystem 330 is complete (as shown) only if the light emitted from the light modulation subsystem does not contain any wavelengths shorter than 495 nm. In practice, if the light coming from the light modulation subsystem 330 contains wavelengths shorter than 495 nm (eg, blue wavelength), some of the light from the light modulation subsystem will pass through the filter 346 and reach the detector 348. do. In such an embodiment, the filter 346 functions to change the balance between the amount of light reaching the detector 348 from the first light source 332 and the amount of light reaching the detector 348 from the second light source 334. do. This can be beneficial if the first light source 332 is significantly stronger than the second light source 334. In another embodiment, the second light source 334 can emit red light and the dichroic filter 346 filters and removes visible light other than red light (eg, visible light having a wavelength shorter than 650 nm). be able to.

Devices and methods for removing one or more micro-objects using optically driven convection and micro-object displacement Micro-objects such as living cells or embryos are not limited to gravity, mechanical pump-guided fluid flow, electro. Several forces, including wetting and / or dielectrophoresis (DEP), can move in a local environment, such as in a microfluidic device. From one location (eg, a particular location where microobjects may have been cultured within a microfluidic device) to another (eg, another area of the same microfluidic device or a separate device such as a multiwell plate) Various force vectors can be applied to achieve cell repositioning in order to move micro-objects more efficiently. Dielectrophoresis (DEP), fluid displacement, etc. may be sufficient to move the cells as desired, but on different scales (eg, stronger or more local forces), different methods (convection forces, etc.). Shear fluid forces, impact forces such as cavitation or air bubble contact with meniscus, or any combination thereof) and / or forces applied on different time scales (eg, durations from a few milliseconds to a few minutes). It can also be utilized to assist in the migration of cells from their current location and / or to selected locations. In a non-limiting example, the application of forces other than DEP may be useful for moving living cells cultured in a microfluidic device over a period of time. The cell may be attached to the surface of the microfluidic device so that DEP force or gravity is not sufficient to move the cell from the attachment position. Thus, a force with other properties, one in which the DEP force is insufficient, or gravity or mechanically pumped fluid flow is unable to selectively and / or sufficiently remove selected cells. Alternatively, it may be useful in removing multiple living cells.

Surprisingly, optical illumination of selected discrete regions on or within the microfluidic device heats a portion of the fluid medium within the microfluidic circuit of the microfluidic device, causing at least a portion of the displaced microobject to The microobjects (including, but not limited to, living cells) are removed and / or the fluid medium (which may include microobjects containing living cells) within the microfluidic device, while still providing viability. It has been found that different scales, physical types, and / or time scales that can be mixed can provide a variety of displacement forces. The generation of such displacement forces can be applied more than once to the same selected discrete region or regions adjacent to it, whereby the forces are repeatedly applied and are sufficiently non-destructive towards micro-objects. While being, cells can be removed and / or media (which may contain microscopic objects) can be mixed. In some embodiments, from one area, which can be a chamber, isolation enclosure, or other microfluidic circuit element of the microfluidic device, to another area and / or location within the microfluidic device, or alternative to the micro. Repositioning cells to another device outside the fluid device (eg, a multiwell plate) can be achieved by applying optical illumination pulses to selected discrete regions within the microfluidic device. Optical illumination pulses can be applied to or in the vicinity of cells of interest. The applied force vector is a function of the energy, duration, and position of the optical illumination pulse. In some embodiments, optical illumination pulses are used to locally heat the surrounding cellular medium (ie, fluid medium), thereby increasing local vapor pressure and creating bubbles at the vapor-fluid interface. Can be generated. The effect of heat-induced bubble formation on the ambient fluid medium and / or cells can vary depending on the duration and configuration of the microfluidic device and / or thermal target. Some types of effects may include:

Cavitation A short light pulse can be used to heat a thermal target and create temporary bubbles. When the bubbles collapse, they generate a cavitation force that can remove potentially nearby cells. In some embodiments, short light pulses are directed at one or more cells, which one or more cells can be removed by the cavitation force thus formed.

Shear Flow In other embodiments, bubbles can grow by continuously illuminating to generate a fluid shear flow directed at nearby cells, thereby removing the cells.

Meniscus contact Alternatively, the bubbles generated by heating the fluid medium at the portion of the thermal target can be directed at the cells. As the bubbles move, the meniscus of the bubbles can contact the cells and remove them from the surface.

In other embodiments, the bubbles can grow until they are thermodynamically preferred to stabilize and persist in the fluid medium. The bubbles can then displace the surrounding liquid phase, thereby removing the cells.

Unintentionally limited by convection theory, the illumination of a thermal target that produces heating of the fluid medium around the thermal target can cause the bubble to have a nucleus and propagate the bubble. The presence of persistent bubbles with a thermal gradient can create thermocytic convection (Gibbs-Marangoni effect). The Gibbs-Marangoni effect refers to the flow of liquid along a surface tension gradient. The liquid can flow from an area with low surface tension to an area with high surface tension. Since the surface tension is lower at higher temperatures, the temperature gradient on the bubble surface allows the liquid around the bubble to flow in the direction of the gradient (ie, from the hot area to the cold area), which can form convection. For the sake of brevity, the flow generated by the temperature gradient over the bubble surface is referred to herein as the "Marangoni effect flow". The greater the temperature difference between the hot and cold areas of the surface, the faster the Marangoni effect flow. The flow can be modulated by changing the optical power. Such convection can be used to move cells or mix fluid media. In some embodiments, a circulating flow generated by heating induced by optical illumination can be used to remove cells within the isolation region of the isolation enclosure. In other embodiments, the circulating flow can be directed to a local region of a microfluidic device that has no fluid flow without the circulating flow, which can be used to mix the medium in the local region.

After removing the cells, further repositioning of the cells can be achieved by using any other suitable cell migration method, including but not limited to fluid displacement, DEP, gravity, etc.

Optical Illumination Optical illumination can be a coherent light source (eg, a laser) or a non-coherent light source. The coherent light source may be a laser characterized by a wavelength in the visible light spectrum (eg, a red wavelength such as 662 nm), or a laser characterized by a wavelength in the infrared portion of the spectrum (eg, a near infrared wavelength such as 785 nm). It may be, or it may be a laser having any other suitable wavelength. The non-coherent light source may include light having wavelengths in the visible range and / or light having wavelengths in the ultraviolet (UV) or infrared range. The light source may provide structured or non-structured light. The temperature gradient induced by illumination with a light source can be modulated by increasing or decreasing the intensity of the light source. Structural light sources can be modulated in several ways to control the properties of structural light sources (eg, using DMDs to spatially modulate the light sources, or using apertures and objectives. Modulate the light source as described above with respect to FIG. 3B.

Without being constrained by theory, incident optical illumination can pass through transparent, substantially transparent, and / or translucent covers or bases of enclosure microfluidic devices. After passing through the cover or base of the enclosure, the incident illumination can be transmitted to a thermal target that is configured to convert the optical illumination into thermal energy, as described below.

Power Non-coherent light can be projected in the range of about 1 milliwatt (mW) to about 1000 milliwatts (mW), but is not limited to this range. In some embodiments, the power of structured or unstructured non-coherent light is from about 1 milliwatt to about 500 milliwatts, from about 1 milliwatt to about 100 milliwatts, from about 1 milliwatt to about 50 milliwatts, from about 1 milliwatt to about 1 milliwatt. 20 milliwatts, about 10 milliwatts to about 500 milliwatts, about 10 milliwatts to about 200 milliwatts, about 10 milliwatts to about 100 milliwatts, about 50 milliwatts to about 800 milliwatts, about 50 milliwatts to about 500 milliwatts, about 50 milliwatts to about 200 milliwatts , About 75 milliwatts to about 700 milliwatts, about 75 milliwatts to about 400 milliwatts, about 75 milliwatts to about 175 milliwatts, or any value in between. Depending on the area where the light is focused and the duration of the illumination, the power of the non-coherent light can be higher or lower than any of the power levels mentioned above. Coherent light can be projected in the range of about 1 milliwatt to about 1000 milliwatts, but is not limited to this range. Depending on the area where the light is focused and the duration of the illumination, the power of the coherent light can be higher or lower than any of the power levels mentioned above. In some embodiments, the power of coherent light is 1 milliwatt to about 500 milliwatts, about 1 milliwatt to about 100 milliwatts, about 1 milliwatt to about 50 milliwatts, about 1 milliwatt to about 20 milliwatts, about 10 milliwatts to about 500 milliwatts. Milliwatts, about 10 milliwatts to about 200 milliwatts, about 10 milliwatts to about 100 milliwatts, about 50 milliwatts to about 800 milliwatts, about 50 milliwatts to about 500 milliwatts, about 50 milliwatts to about 200 milliwatts, about 75 milliwatts to about 700 milliwatts, It can be in the range of about 75 milliwatts to about 400 milliwatts, about 75 milliwatts to about 175 milliwatts, or any value in between.

The power of the incident light can be chosen differently based on the type of removal force desired. For example, if a circulating flow that can incorporate the Marangoni effect flow is desired, the power of the incident light can be selected to be as low as 1 milliwatt and variously modulated when the circulating flow is established and / or maintained. Can be done. When removing micro-objects by using cavitation force, shear flow force, or bubble contact force, the power can be selected to be in a higher range, eg, about 10 milliwatts to about 100 milliwatts. The power can also be adjusted based on the desired duration of lighting.

Illumination Location The illumination location can be selected to be any selected discrete region of the microfluidic device, as it may be useful. In some embodiments, the selected discrete region of illumination can be a location within the isolation enclosure of the microfluidic device. In various embodiments, the selected discrete region of illumination is located within the isolation region of the isolation enclosure, and the isolation enclosure is not limited to 124, 126, 128, 130, 224, 226, 228, 266, 502. , 504, 506, 604, 606, 608, 704, 732, 734, 736, 738, 802, 804, 806, 902, 1002, 1102, 1202, 1402, 1502, as described herein. It can be configured like an isolation enclosure. In various embodiments, the selected discrete region of illumination can be within the displacement force generation region of the isolation enclosure, as more fully described below. In other embodiments, the discrete regions of illumination can be within the circulating culture enclosure. If the illumination is performed within the circulation culture enclosure, the illumination may be directed to the microfluidic channel at the displacement force generation region, the connection region, the selected discrete region within the cell culture region or the opening of the circulation culture enclosure. In yet another embodiment, the selected discrete region of illumination may be located more completely within the microfluidic channel, as described below.

Microfluidic Devices The present disclosure provides microfluidic devices configured to be capable of optically driven convection generation and / or displacement of internal microobjects. In one aspect of the disclosure, a microfluidic device with an enclosure is provided, the enclosure may include a flow region and an isolation enclosure, the isolation enclosure may include a connection region and a isolation region, the connection region being the proximal end to the flow region. Includes an opening and a tip opening to a separation area. The isolation enclosure may include a thermal target in the isolation area. In various embodiments, the isolation enclosure further comprises a displacement force generation region, the separation region comprises at least one fluid connection to the displacement force generation region, and the displacement force generation region further comprises a thermal target. In various embodiments, the microfluidic device may have at least one isolation enclosure, which is isolation enclosure 124, 126, 128, 130, 224, 226, 228, 266, 502, 504, 506, 604. , 606, 608, 704, 732, 734, 736, 738, 802, 804, 806, 902, 1002, 1102, 1202, 1402, 1502. In various embodiments, the thermal target or displacement force generating region may be configured to limit the expansion of bubbles formed therein in one main direction.

In various embodiments, the enclosure of the microfluidic device may further include a cover that partially defines the isolation enclosure, and the thermal target may be placed on the cover. In some embodiments, the thermal target may be placed on the inner surface of the cover facing the enclosure. In other embodiments, the enclosure of the microfluidic device may further include a microfluidic circuit structure that partially defines the isolation enclosure, and the thermal target may be placed in the microfluidic circuit structure. In yet another embodiment, the enclosure of the microfluidic device may further include a base that partially defines the isolation enclosure, and the thermal target may be located on the inner surface of the base.

In another aspect of the disclosure, a microfluidic device with an enclosure is provided, wherein the enclosure is a microfluidic circuit configured to include a fluid medium to accommodate at least one circulating flow of the fluid medium. A microfluidic circuit that is constructed and a first thermal target located on the surface of an enclosure within the microfluidic circuit that, when optically illuminated, produces a first circulating flow of the fluid medium. Consists of a first thermal target configured in.

In various embodiments of the microfluidic device, including microfluidic circuits configured to accommodate circulating flow, the enclosure of the microfluidic device may further include a microfluidic channel and an isolation enclosure, and the isolation enclosure is a microfluidic. Adjacent to the channel, it can open towards the microfluidic channel. In various embodiments, the isolation enclosure is the isolation enclosure 124, 126, 128, 130, 224, 226, 228, 266, 502, 504, 506, 604, 606, 608, 704, 732, 734, 736, 738, It can be configured as any of 802, 804, 806, 902, 1002, 1102, 1202, 1402, 1502. In other embodiments, the microfluidic device, the enclosure of the microfluidic device, including the microfluidic circuit configured to correspond to the circulating flow, may further include a microfluidic channel and a circulating culture enclosure. The circulation culture enclosure can be configured as any of the circulation culture enclosures 602, 802, 1302. The circulating culture enclosure may open in the microfluidic channel and may further have any other features or dimensions as described herein for the circulating culture enclosure.

In various embodiments of a microfluidic device, including a microfluidic circuit configured to correspond to a circulation flow, the circulation flow path may include part of a channel and at least part of an isolation enclosure. In other embodiments, the circulation channel may be located within the isolation enclosure. In some embodiments, the circulation channel may include a constriction.

In various embodiments of a microfluidic device, including a microfluidic circuit configured to correspond to a circulating flow, the microfluidic device is optically illuminated to generate a second circulating flow of the fluid medium. Can include a second thermal target configured in. The second thermal target may be placed adjacent to the first thermal target on the surface of the enclosure. The second thermal target may be located in the same microfluidic circuit as the first thermal target. In various embodiments, the first thermal target and the second thermal target can be directed to provide a first circulating flow and a second circulating flow of the fluid medium in opposite directions.

In various embodiments of the microfluidic device, including microfluidic circuits configured to accommodate circulating flow, the thermal target is located on a surface within the microfluidic channel. In some embodiments, the enclosure of the microfluidic device may further comprise more than one microfluidic channel, the first microfluidic channel being the second in a first position along the second microfluidic channel. It can be configured to open from the microfluidic channel of the, reconnect to the second microfluidic channel in the second position, and thereby further configured to form a microfluidic circuit, the thermal target being the first. Can be placed on the surface within the microfluidic channel of. In various embodiments, at least one isolation enclosure can be opened in the first microfluidic channel. In various embodiments, at least one isolation enclosure is the isolation enclosure 124, 126, 128, 130, 224, 226, 228, 266, 502, 504, 506, 604, 606, 608, 704, 732, 734, 736. , 738, 802, 804, 806, 902, 1002, 1102, 1202, 1402, 1502. In some embodiments, the fluid resistance of the first channel can be about 10 to about 100 times the fluid resistance of the second channel. The second microfluidic channel can have a width about 1.5 to about 3 times greater than the width of the first microfluidic channel. In some embodiments, the width of the second microfluidic channel is from about 100 μm to about 1000 μm. In various embodiments, the width of the first microfluidic channel can be from about 20 μm to about 300 μm.

In yet another aspect, a microfluidic device with an enclosure is provided, the enclosure comprising a microfluidic channel and an isolation enclosure, in which the isolation enclosure is adjacent to the microfluidic channel and opens in the microfluidic channel, providing a thermal target. Located in a channel adjacent to the opening to the isolation enclosure, the thermal target is further configured to direct the flow of the fluid medium towards the isolation enclosure when optically illuminated. In various embodiments, at least one isolation enclosure is the isolation enclosure 124, 126, 128, 130, 224, 226, 228, 266, 502, 504, 506, 604, 606, 608, 704, 732, 734, 736. , 738, 802, 804, 806, 902, 1002, 1102, 1202, 1402, 1502. In some embodiments, microfluidic devices having isolation enclosures and thermal targets within the channel are as isolation enclosures 502, 504, 506, 602, 604, 606, 608, 704, 732, 734, 736, 738, 902. If it has a configured quarantine enclosure, the quarantine enclosure does not have to have a thermal target within the quarantine enclosure itself. In some embodiments, the thermal target can be placed on a surface within the microfluidic channel.

For any microfluidic device, the enclosure may further include a dielectrophoretic configuration. In some embodiments, the dielectrophoretic configuration can be optically actuated.

In any microfluidic device, at least one surface of the enclosure may include a covering surface. In some embodiments, the isolation enclosure of the microfluidic device may include at least one surface that is a covering surface. In some embodiments, the covering surface can be a covalently modified surface.

Thermal Target A thermal target is a microfluidic feature of a microfluidic device that can be a distinct feature designed for this purpose. Alternatively, the thermal target can be a location within the microfluidic circuit to which optical illumination is applied. The thermal target is a passive microfluidic feature and does not include any self-activating resistance or electric heater. The passive nature of the thermal target simplifies the fabrication of microfluidic devices. For thermal targets containing metals or microstructures, the complexity of fabrication is much lower than for active thermal targets such as resistors, as described below. Unlike the passive thermal targets of the present disclosure, active thermal targets such as resistors must have a fixed electrical connection and are made in a fixed position. When the thermal target is in the selected position of the microfluidic circuit material or base, the flexibility to generate force specifically selectively where it is needed, without additional structural features, is fixed with the active thermal target. It is particularly advantageous in comparison.

4A-4E show the geometry of various thermal targets according to some embodiments of the present disclosure. As will be appreciated by those skilled in the art, any of the properties of the thermal targets shown in FIGS. 4A-4E can be combined to produce thermal targets with a variety of desired functions.

FIG. 4A shows a square thermal target 430. The blunt angles and uniform sides of the thermal target 430 of FIG. 4A can be advantageous in nucleating substantially uniform bubbles. Similarly, the circular heat target 432 shown in FIG. 4B also provides a shape configured to generate a uniform local heat source and nucleate a substantially uniform bubble.

Conversely, the thermal targets shown in FIGS. 4C, 4D, 4E, and 4G have an asymmetric shape, which can be advantageous for the generation of bubbles with a temperature gradient. Unintentionally limited by theory, bubbles with a temperature gradient can produce the Gibbs-Marangoni effect (also known as thermocytic convection), as described above. The asymmetric thermal target 434 shown in FIG. 4C features a teardrop-like shape used to generate bubbles with a temperature gradient that can be used to generate the Marangoni effect flow. Since the teardrop-shaped wide portion 434a has a larger surface area than the teardrop-shaped tapered portion 434b, it produces a higher temperature when heated by optical illumination. Therefore, in the bubbles generated using the asymmetric thermal target 434 of FIG. 4C, the area of the bubbles located over the wide portion 434a of the thermal target has a higher temperature than the area of the bubbles located over the tapered portion 434b of the thermal target. Can have a temperature gradient to have.

The temperature gradient can also be modulated by physically separated, differently sized parts of the thermal target. FIG. 4D shows a thermal target 436 composed of two parts 438 and 440 of different sizes, which are physically separated but located close together so that they can be heated using the same structural light source. The physical distance between the two parts 438 and 440 of the thermal target 436 creates a larger temperature difference that can be used to generate a faster Marangoni effect flow, thus increasing the force. FIG. 4E shows a thermal target 442 further divided into three parts 444, 446, 448.

The thermal targets 430, 432, 434, 436, 442, 450, 452 shown in FIGS. 4A-4G are formed by depositing a continuous or discontinuous metal shape on one surface of the microfluidic circuit structure 108 or cover 110. Can be made. In some embodiments, the thermal target can be deposited on the inner surface of the cover 110. The thermal target faces the interior of the enclosure 102 (chamber / region 202) where it may come into contact with the fluid medium. Thermal targets 430, 432, 434, 436, 442, 450, 452 can include any type of metal that can be excited by a light source to generate heat. Suitable metals include chromium, gold, silver, aluminum, indium tin oxide, or any combination thereof. Other metals (and alloys) are also known in the art. The thermal target 440 can have a continuous metal surface or can be made of a discontinuous shaped metal (eg, a metallic shape such as a dot). Various patterns can be used to optimize the heating and formation of uniform bubbles.

FIG. 4F shows a thermal target 450 containing a discontinuous metal shape. In the embodiment shown in FIG. 4F, the shape is a dot. However, any type of metal shape can be used (eg squares, lines, cones, wavy lines). In addition, a variety of different metal shapes can be used with the same thermal target.

In some embodiments, the discontinuous metal shape can be distributed at ever-increasing concentrations to enhance the temperature gradient of the thermal target 450. FIG. 4G shows a patterned thermal target 452 with a gradient of metal shape. The temperature gradient of the thermal target 452 can be strengthened by increasing the distribution density of the metal dots in the wide portion 452a of the thermal target 452 and decreasing the distribution density of the metal dots in the narrow portion 452b of the thermal target 452.

In some embodiments, the thickness of the metal deposited on the thermal target as a continuous or discontinuous metal shape can be varied to enhance the temperature gradient of the thermal target. For example, thicker metal deposits can be used to generate higher heat in the larger (or wider) portion of the thermal target, thereby enhancing the temperature gradient. In some embodiments, the thickness of the metal deposited on the thermal target ranges from about 3 nm to about 50 nm, about 3 nm to about 30 nm, about 5 nm to about 50 nm, about 5 nm to about 30 nm, and about 5 nm to about 25 nm. Or it can be any value between them.

Microstructure As mentioned above, in some embodiments, the microfluidic circuit structure 108 or cover 110 of the enclosure 102 facilitates bubble nucleation and / or formation from the heat generated when optically illuminated. And may have one or more microstructures introduced to create surface topography that can function as a thermal target. Microstructures can be discontinuous Microstructures can be negative features (eg, depressions or depressions made on the surface of the base or the surface of the wall. As is known in the art, pillars, dots, cavities. , Or a microstructure such as a depression can be patterned into a microfluidic circuit structure 108 or cover 110 to create a location suitable for bubble nucleation. FIG. 4H shows a thermal target 454 containing a depression that can be used for bubble nucleation. It is a stylized figure. In some embodiments, surface topography can be combined with metal patterns in various ways to create an ideal surface for heat absorption that subsequently produces displacement forces. Microstructures. When viewed from above, in the x-axis and y-axis directions, it exceeds the range of about 50 square μm, 100 square μm, about 200 square μm, about 300 square μm, about 500 square μm, or any value between them. It may have an area. The microstructure may include only one unit or together may be a plurality of microstructures having the total area described above. Negative microstructures are placed on the base. It can be formed by focusing a light source (eg, laser or non-coherent light) on a patternable microfluidic circuit material that can or can be part of a wall, and the focused light is a patternable microfluidic circuit material. Can be patterned to form depressions or depressions.

Alternatively, the microstructure can rise above the surface of the base, for example, or extend from the walls of the enclosure, flow area, or isolation enclosure (as a non-limiting example, pillars or dots). (Not shown). The microstructure can have any convenient fabrication height within the enclosure. The microstructure can still have a height that allows microobjects such as living cells to pass through the microstructure. The height of the microstructure can be about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, or any value between them. Each of the plurality of microstructures does not have to have the same height and may have different heights from each other. Positive microstructures can be formed from the same materials used to form the microfluidic circuit structure, such as PDMS or any photopatternable silicone, microfluidic circuits such as walls, isolation enclosures, or channels. It can be formed during the same process as that used to make other elements.

In some other embodiments, the positive microstructure can be formed from hydrogels such as photoinitiator polymers. The photoinitiator polymer can be a synthetic polymer, a modified synthetic polymer, or a photoactivated biopolymer. In some embodiments, the biopolymer can be modified to incorporate a moiety that provides a photoactivating ability.

In some embodiments, the photoinitiator polymer is polyethylene glycol, modified polyethylene glycol, polylactic acid (PLA), modified polylactic acid, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide. , Poly-N-isopropylacrylamide (PNIPAm), modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, polycaprolactone (PCL), modified polycaprolactone , Fibronectin, Modified Fibronectin, Collagen, Modified Collagen, Laminine, Modified Laminine, Polysaccharide, Modified Polysaccharide, or at least one of any combination of copolymers. In other embodiments, the polymer is polyethylene glycol, modified polyethylene glycol, polylactic acid (PLA), modified polylactic acid, polyglycolic acid (PGA), modified polyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacryl. At least of acid (PAA), modified polyacrylic acid, polycaprolactone (PCL), modified polycaprolactone, fibronectin, modified fibronectin, collagen, modified collagen, laminin, modified laminin, polysaccharide, modified polysaccharide, or any combination of polymers. Can include one. In yet another embodiment, the polymer is polyethylene glycol, modified polyethylene glycol, polylactic acid (PLA), modified polylactic acid, polyglycolic acid (PGA), modified polyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol, poly. It may include at least one of acrylic acid (PAA), modified polyacrylic acid, fibronectin, modified fibronectin, collagen, modified collagen, laminin, modified laminin, or any combination of polymers. In some embodiments, the photoinitiator polymer does not include a silicone polymer. In some embodiments, the photoinitiator polymer may be free of polylactic acid (PLA) or modified polylactic acid polymer. In other embodiments, the photoinitiator polymer may be free of polyglycolic acid (PGA) or modified polyglycolic acid polymer. In some embodiments, the photoinitiator polymer may be free of polyacrylamide or modified polyacrylamide polymer. In yet other embodiments, the photoinitiator polymer may be free of polyvinyl alcohol (PVA) or modified polyvinyl alcohol polymer. In some embodiments, the photoinitiator polymer may be free of polyacrylic acid (PAA) or modified polyacrylic acid polymer. In some other embodiments, the photoinitiator polymer may be free of polycaprolactone (PCL) or modified polycaprolactone polymer. In other embodiments, the photoinitiator polymer does not have to be formed from fibronectin or modified fibronectin polymer. In some other embodiments, the photoinitiator polymer does not have to be formed from collagen or modified collagen polymer. In some other embodiments, the photoinitiator polymer need not be formed from laminin or modified laminin polymer.

The physical and chemical properties that determine the stability of the polymer used in the solidified polymer network include molecular weight, hydrophobicity, solubility, diffusion rate, viscosity (eg, of the medium), excitation, and / or radiation range (eg, of the medium). , Of internally immobilized fluorescent reagents), known background fluorescence, properties that affect polymerization, and pore size of the solidified polymer network. The photoinitiator polymer can be formed by polymerizing a fluid polymer (eg, a prepolymer solution). Briefly, the fluid polymer solution can flow into a microfluidic device and solidify in situ before being used in the methods described herein. The method of installing microstructures derived from photoinitiating polymers is fully described in US Patent Application No. 15/372094 filed December 7, 2016.

Among the many polymers that can be used, one type of polymer is polyethylene glycol diacryllate (PEGDA). Photoinitiated polymerization is very efficient, is a non-yellowing radical, and can usually be initiated in the presence of the free radical initiator Igracure® 2959 (BASF), which is an α-hydroxyketone photoinitiator. , UV region (eg, 365 nm) wavelengths are used for initiation, but other initiators can also be used. An example of another photoinitiator class useful for polymerization reactions is the group of lithium acyl phosphinate salts, the lithium phenyl 2,4,6, -trimethylbenzoyl phosphinate, which is α. It has certain utility due to its more efficient absorption at wavelengths longer than the hydroxyketone class (eg, 405 nm).

Other types of PEG that can be photopolymerized include PEG dimethylacryllate and / or multi-arm PEG (n-PEG) acrylate (n-PEG-Acr). Other polymer classes that may be used include polyvinyl alcohol (PVA), polylactic acid (PLA), polyacrylic acid (PAA), polyacrylamide (PAM), polyglycolic acid (PGA), or polycaprolactone (PCL). Be done.

The molecular weight range of the polymer can vary depending on the performance needs of the microstructure. Depending on the structure of the polymer, a fluid polymer with a wide range of molecular weights may be suitable. Useful star polymers are Mw (weight average molecular weight) in the range of about 500 Da to about 20 kDa (eg, 4-arm polymers), or up to about 5 kDa for each arm or linear polymer, or any value between them. Can have. In some embodiments, polymers with higher molecular weight ranges may be used in fluidized polymers at lower concentrations and may nevertheless provide microstructures that can be used in the methods described herein.

Various copolymer classes can be used, including but not limited to the polymers listed above or biopolymers such as fibronectin, collagen, or laminin. Polysaccharides such as dextran or modified collagen can be used.

In some embodiments, the microstructure can be part of, or part of, the sacrificial features used in the methods described herein, absorb optical illumination, and move microobjects. Can be deformed or degraded as a result of producing a thermal effect that can be used in.

Alternatively, the thermal target is in by projecting structural light onto the microfluidic circuit structure 108 or cover 110 to create an optical pattern with the same geometry as the thermal target 450.
It can be made in situ. This technique does not require any special metal deposition or microfluidic circuit material patterning. FIG. 4I provides a stylized view of the thermal target 456 made by projecting a circular pattern of light onto the microfluidic circuit structure 108 or cover 110. It can also be used in conjunction with a thermal target that projects a pattern of light with a particular geometry, including various geometries, surface topography, metal patterns, and combinations thereof.

In yet another embodiment, the optical illumination is focused on a portion of the microfluidic circuit material on the wall of the isolation enclosure, the inner surface of the base facing the enclosure of the microfluidic device, or a selected point on the microfluidic channel wall. It does not include any of the special features mentioned above for thermal targets. However, these selected discrete regions of isolation enclosure or wall microfluidic circuit material or the inner surface of the base can also be used as thermal targets and can serve as sacrificial features.

Size Thermal targets are about 1 mm, about 0.9 mm, about 0.7 mm, about 0.5 mm, about 0.3 mm, about 100 μm, about 80 μm, about 60 μm, about 40 μm, about 20 μm, about 10 μm, about 5 μm, or It may have a first dimension of any value between them (eg, the width of the microfluidic enclosure or the x-axis dimension). The steps of illuminating the selected discrete region are about 1 mm, about 0.9 mm, about 0.7 mm, about 0.5 mm, about 0.3 mm, about 100 μm, about 80 μm, about 60 μm, about 40 μm, about 20 μm, about. It may further include illuminating an area having a second dimension of 10 μm, about 5 μm, or any value between them (eg, the y-axis dimension within the microfluidic enclosure). The x-axis dimension or the y-axis dimension can be any combination of the above dimensions. In some embodiments, the thermal target may have an x-axis dimension of about 100 μm and a y-axis dimension of about 100 μm. In other non-limiting embodiments, the thermal target may have an x-axis dimension of about 5 μm and a y-axis dimension of about 5 μm.

Some Embodiments of Optically Driven Convection and Displacement Isolation Enclosures and Circulation Culture Enclosures As mentioned above, isolation enclosures useful for optically driven convection and / or displacement of microscopic objects are fluidly connected to the isolation region of the isolation enclosure. It is possible to have a displacement force generation region to be generated, and a minute object can be arranged in the displacement force generation region and maintained arbitrarily. The displacement force generation region can be connected to the tip of the separation region opposite to the opening of the separation region to the connection region. Alternatively, the displacement force generation region may connect to the separation zone at or adjacent to the opening of the separation zone to the connection zone. In some embodiments, the displacement force generating region may have two or more fluid connections to the separation region (see FIGS. 7A-7F). In some embodiments, the isolation enclosure may include a circulation channel. The circulation flow path may include a separation region and a displacement force generation region (see FIGS. 6A-6C). In some embodiments, the circulation channel may include a constriction (see FIG. 6A).

In some other embodiments, the displacement force generation region may include one or more fluid connections to the separation zone that interfere with the opening of the separation zone to the connection zone. In some embodiments, at least one fluid connection between the separation region and the displacement force generation region may include cross-sectional dimensions configured to prevent micro-objects from passing from the separation region to the displacement force generation region. Damage from heat or impact can be reduced by blocking possible entry of the micro-object into the displacement force generating region, further assisting the maintenance of the micro-object within the isolation region of the isolation enclosure. In some embodiments, at least one fluid connection between the separation region and the displacement force generation region comprises one or more barrier modules (FIGS. 7A-7F, 726, 726a, 726b, 726c, 726d). , Or see 726e), one or more barrier modules are configured to prevent micro-objects from passing from the separation region to the displacement force generation region. The barrier module can be of any size or shape, the gap between the barrier module and the adjacent barrier module (see FIGS. 7A-7F, 728, 728a, 728b, 728c, 728d, 728e) or the barrier module. The gap between and the wall of the isolation enclosure can have dimensions that prevent microscopic objects such as living cells from passing from the separation region to the displacement force generation region. In some embodiments, microobjects such as microbeads can pass from the separation region to the displacement force generation region, but microobjects such as living cells or embryos do not pass into the displacement force generation region due to the barrier module. The barrier module measures the dimensions across the isolation enclosure, which is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, about 80% of the width of the isolation enclosure, or any value between them. Can have. The gap between the barrier module and the adjacent barrier module or the gap between the barrier module and the wall is about 5 μm, about 7 μm, about 9 μm, about, depending on the size of the micro-objects placed in the separation area. It can be 11 μm, about 13 μm, about 15 μm, about 17 μm, about 20 μm, about 25 μm, or about 40 about μm.

In various embodiments, at least one fluid connection between the separation region and the displacement force generation region, except due to diffusion, provides fluid flow from the displacement force generation region in the absence of any internally generated force. It may have a cross-sectional dimension configured to block. (See FIGS. 9A-9C). The dimensions of the displacement force generation region can match the dimensions of the separation region. The displacement force generation region can be a separate compartment of the isolation enclosure. In various embodiments, the displacement force generation region can be configured to minimize secondary flow.

In some embodiments, the isolation enclosure may include a second thermal target configured to generate a second circulation flow of the fluid medium when optically illuminated. The second thermal target can be placed within the displacement force generation region. The first thermal target and the second thermal target can be directed to provide a first circulation flow and a second circulation flow of the fluid medium in the opposite direction.

In various embodiments, the displacement force generating region may include one opening, which may be a fluid connection of the displacement force generating region to the separation region. In some embodiments, the fluid connection in the displacement force generating region may include a fluid connector. (See fluid connector 514 in FIG. 5A). In some embodiments, the fluid connector in the displacement force generation region may include at least one bend (see fluid connector 514 in FIG. 5A). In some embodiments, at least one bend of the fluid connector is about 60 degrees to about 180 degrees, about 60 degrees to about 120 degrees, about 60 degrees to about 90 degrees, about 40 degrees to about 180 degrees, about. It may include turns of 40 degrees to about 120 degrees, about 40 degrees to about 90 degrees, or any value between them. In other embodiments, the fluid connector in the displacement force generating region may include at least two bends. In some embodiments, each of the at least two bends of the fluid connector is about 60 degrees to about 180 degrees, about 60 degrees to about 120 degrees, about 60 degrees to about 90 degrees, about 40 degrees to about 180 degrees. , About 40 degrees to about 120 degrees, about 40 degrees to about 90 degrees, or any value of turns between them. If the turn is contained in the fluid connector between the displacement force generation area and the separation area, the isolation enclosure as a whole resembles a "U", "N", or inverted "N" shape. Can have.

In various embodiments, the width of the fluid connector can be the same as the width of the separation region and / or the displacement force generation region. In some embodiments, the fluid connector may include cross-sectional dimensions in the x-axis and y-axis dimensions that are configured to prevent micro-objects from passing from the separation region to the displacement force generation region. In various embodiments, the height of the fluid connector in the z-axis direction can vary so that microobjects cannot pass through. From the separation area to the displacement force generation area.

In various embodiments, the thermal target or displacement force generating region within the isolation enclosure may be configured to limit the expansion of formed bubbles in one primary direction. For example, the displacement region can be elongated and is located at the tip of the displacement region so that cavitation force / bubble growth / shear flow / convection is forced in the direction towards the fluid connection with the separation region. It may have a thermal target. This is not a description that limits the configuration of the thermal target or displacement force generation region that can limit the expansion of bubbles, and other configurations are possible.

In various embodiments, the thermal target may be positioned at a portion of the displacement force generating region at the tip to at least one fluid connection to the separation region. In some embodiments, the displacement force generation region can have a width of about 20 μm to about 100 μm in the x-axis or y-axis dimensions, depending on the orientation of the displacement force generation region within the microfluidic device. In some embodiments, the displacement force generating region can be configured to minimize the secondary flow of the fluid medium, thereby maximizing the force directed at the cells within the separation region.

The circulating culture enclosure may have a contiguous zone and a displacement force generating zone having any combination of the features or dimensions described above for the isolation enclosure. The circulating culture enclosure can have any size or feature described for the isolation region of the isolation enclosure, but when actively circulating, the circulating culture enclosure circulates the flow through the main channel into the culture region. Can include different culture areas in that they are composed of. In various embodiments, the displacement force generating region of the circulating culture enclosure may further include an opening to the flow region. One embodiment of the circulation culture enclosure will be described more fully below with respect to FIG. 6A.

The configuration and use of the microfluidic devices of the present disclosure can be more fully understood by reference to FIGS. 5A-8D.

FIG. 5A shows an example of an isolation enclosure 502 that opens towards channel 522 configured to include fluid medium flow 530 in the microfluidic device 500. The isolation enclosure 502 includes a thermal target 540 configured to generate bubbles (not shown) used to eject the microobject 504 from the isolation enclosure 502, according to some embodiments of the present disclosure. Isolation enclosure 502 includes isolation region 510 for storing and / or culturing microscopic objects 504 such as cells. The separation region 510 and the thermal target 540 are physically separated by the displacement force generation region 512, and the displacement force generation region 512 is nucleated by focusing light (not shown) on the thermal target 540. Also included is a fluid connector 514 that allows sufficient space to increase volume (referred to herein as "bubble expansion"). As the bubbles grow due to the increase in volume, the expanding bubbles generate a force against the fluid in the displacement force generation region (512 + 514), thus creating a shear flow (not shown) of the fluid along the path 516. In many embodiments, the thermal target 540 can be positioned at the tip of the displacement force generation region 512 (+514) to ensure that it exerts a force in the main direction as the bubble expands. In some embodiments, either the thermal target or the displacement force generating region expands the bubble so that it can expand in only one main direction when thermal energy continues to be supplied by continuous illumination. Is configured to limit. The fluid connector 514 between the separation region and the displacement force generation region (512 + 514) blocks the fluid flow from the displacement force generation region (512 + 514) in the absence of any internally generated force, except for the force due to diffusion. It may have a cross-sectional dimension configured as such.

When the micro-object is a biological micro-object (eg, a living cell), the displacement force generating region (512 + 514) physically physicals the biological micro-object from the heat generated by the thermal target 450 when optically illuminated. It also works to separate the objects. In some cases, the expanding bubble also functions to provide a physical barrier between the microobject 504 and the thermal target 540. As described below, the geometry of the isolation enclosure 502 can be optimized to maximize the force (and the resulting shear flow) from the nucleation and expansion of the bubbles.

The isolation enclosure 502 shown in FIG. 5A has a shape similar to the inverted letter "N" (ie, a shape like the inverted "N"), while other embodiments are from the isolation enclosure 502. It may have different shapes that are beneficial in generating sufficient shear flow to displace the microobject 504.

Further, for brevity, the isolation enclosure 502 collects features or micro-objects 504 used to hold cells in place by the force of gravity and positions the micro-objects 504 within isolation enclosure 502. It is shown without any other features that could actually be used to provide other desirable functions, such as a trap located opposite the quarantine enclosure 502. However, in practice, any other feature described herein for these features or isolation enclosures can be used in conjunction with isolation enclosure 502. Similarly, the isolation enclosure 502 shown in FIG. 5A is shown with a square thermal target 540 used to generate uniform bubbles. In other embodiments, symmetrical thermal targets of other shapes or materials may be used. In yet another embodiment, the thermal target 540 is absent and the optical illumination is directed at the microfluidic circuit material 506 in the vicinity of the thermal target 540 to nucleate bubbles that can be unstable or stable. , A cavitation force that removes microobjects 504, a fluid force of shear flow, or a bubble contact force can be generated.

FIG. 5B shows the formation of bubble 520 and the use of shear flow 542 generated by the growing bubble 520, which displaces the microobject 504 from the isolation enclosure 502 of FIG. 5A. The light source (not shown) focuses on the thermal target 540 and excites (ie, heats) the thermal target 540, thereby nucleating the bubble 520a. By continuing to focus the light source on the thermal target 540, the bubbles 520a can expand in volume to continuously generate larger bubbles 520b, 520c, 520d, 520e. The increased size of the bubble 520 creates a force on the fluid medium (not shown) in the isolation enclosure 502, which creates a shear flow 542 that displaces the microobject 504 from the separation region 510 to the channel 522. When the micro-object 504 is displaced to the channel 522, the micro-object 504 can be manipulated or moved by controlling the flow 530 in the channel 522, or it can be moved using the DEP.

The displacement force generation region 512 includes the fluid connector 514 and can be optimized to reinforce the shear flow 542 by adjusting the geometry and length of the displacement force generation region. For example, the length and width of the displacement force generating region between the thermal target 540 and the separation region 510 can be optimized to generate shear flow 542. The thermal target 540 can be positioned at the tip of the isolation enclosure 502 to ensure that the bubble 520 expands in one direction. Similarly, the width of the tip portion of the displacement force generating region 512 + 514 including the thermal target 540 can be narrowed to ensure that the nucleated bubble 520 expands primarily in one direction. The suitable width of the tip portion of the displacement force generation region 512 + 514 can be in the range of about 20 μm to about 100 μm. In some embodiments, the fluid connector region 514 of the displacement force generation region may have the same width as the tip portion of the displacement force generation region 512.

In some embodiments, the displacement force generation region 512 + 514 can be optimized to minimize secondary flow that can interfere with the shear flow 542. As will be described later, the displacement force generation region 512 + 514 may include a constricted portion in which the width of the displacement force generation region 514 + 512 is significantly reduced. Depending on the embodiment, the width of the constriction portion can be 1/2 to 1/20 of the width of the displacement force generation region 512 + 514. For example, the constriction can have a width in the range of about 5 μm to about 50 μm, and the displacement force generation region 512 + 514 can have a width in the range of about 20 μm to about 100 μm. In some embodiments, the displacement force generating region 512 + 514 comprises one or more (2, 3, 4, or 5) turns in the fluid connector region 514 of the displacement force generating region.

In the case shown in FIG. 5B, the bubble 520 does not contact the micro object 504 and the micro object 650 only receives the shear flow 542 generated by the growth of the bubble. However, in another embodiment of the use of the isolation enclosure 502, the meniscus of the bubble 520 is brought into contact with the microobject 504 to displace the microobject 504 from the separation region 510 and optionally displace the microobject 504 from the isolation enclosure 502. It may be desirable or even advantageous to provide a contact force to carry out. The unloading may be an active unloading driven by the flow of air bubbles, or simply removing the cells so that the cells 504 can be evacuated from the isolation enclosure 502 by another force such as DEP. In some embodiments, the length of the displacement force generating region 512 + 514 may be shortened in order to bring the meniscus of the bubble 520 into contact with the micro-object 504 within the separation region 510. Depending on the embodiment, the displacement force generation region 512 + 514 may partially overlap the separation region 510 and may not even have a fluid connector 514 that includes any turn.

In other cases, it may be advantageous to move through isolation enclosure 502 and nucleate bubble 520 into channel 522. In some cases, bubbles 520 can be used to carry microscopic objects into channel 522. In other cases, bubbles 520 are used to block channel 522 (eg, prevent microobjects from moving through channel 522) and / or flow fluid medium (not shown) flow 530 in channel 522. Can be redirected. For example, in a microfluidic circuit including a plurality of channels 122 (such as the microfluidic circuit 280 shown in FIG. 2F), the bubble 520 is guided and the flow path 106 is redirected from the first channel 122 to one of the other channels 122. Can be used as a blocking mechanism.

Alternatively, this configuration can be used to implement any mode in which the microobject 504 is removed by bubble flow, shear flow, contact of the bubble with the meniscus, or convection.

Other methods and techniques may be used in conjunction with the optical drive displacement and unloading of micro-objects from the isolation enclosure to the channel or other circuit elements. For example, tilting device 190 can be used to tilt (ie, rotate the microfluidic circuit on the horizontal axis) or invert the microfluidic circuit, thereby causing the microobject to receive gravity, which is optically driven. It can be used at the same time as using the method or as a preliminary step thereof. Similarly, in some cases magnetic beads can be used to obstruct or remove microscopic objects. In these cases, the magnetic beads can be placed in the isolation enclosure and removed using magnetic force. The movement of the magnetic beads as they are removed from the isolation enclosure can assist in the displacement and / or removal of microscopic objects attached to the isolation enclosure.

FIG. 5C shows the use of optical illumination directed at the thermal target 541, which can simply be used to generate bubbles 521 within the isolation enclosure 502 of the microfluidic device 500, displacement force generation. A selected discrete region on the inner surface of the region. The selected discrete regions forming the thermal target 541 do not require any metal deposition or any special patterning of the inner surface of the microfluidic circuit material or base. In the embodiment shown in FIG. 5C, the light source may be focused on the area of isolation enclosure 502 with a square pattern of light (not shown). This square pattern of light can heat the microfluidic circuit structure 108, the inner surface 109, and / or the cover 110, thereby creating a thermal target 541 at any selected location, which the thermal target 541 It can be used to nucleate and grow bubbles 521 that generate a shear force 542 sufficient to carry the microobject 504 out of the isolation enclosure 502. Alternatively, this configuration can be used to implement any method of removing microobjects 504 by bubble flow, shear flow, contact of bubbles with meniscus, or cavitation forces.

FIG. 5D shows the isolation enclosure 544 of the microfluidic device 550, in which the repeating numbered elements are defined as described above. The isolation enclosure 544 is configured to generate an optical drive displacement force according to some embodiments of the present disclosure and is used to carry out a microobject 504. The isolation enclosure 544 shown in FIG. 5D is characterized by a shape resembling the letter "U" (ie, a "U" -like shape), with the separation region 554 just below the proximal opening 534 and the contiguous zone 552. It is in. The thermal target 543 is located at the tip of the isolation enclosure 544 within the displacement force generation region 556. Similar to FIG. 5A, the displacement force generation region 556 is a bubble (not shown) nucleation and cavitation force, shearing force, a bubble that can come into contact with the micro object 504, or a micro object 504 within the separation region 554. A sufficient distance between the separation region 554 and the thermal target 543 to create a stream of bubbles that can be removed and optionally allow the use of bubbles to displace the microobject 504 into channel 522. To provide. The path of air bubbles, shear flow, or cavitation force is indicated by path 546.

FIG. 5E shows the isolation enclosure 548 of the microfluidic device 560, in which the repeating numbered elements are defined as described above. The isolation enclosure 548 is configured to generate an optical drive displacement force according to some embodiments of the present disclosure and is used to carry out a microobject 504. The isolation enclosure 548 shown in FIG. 5E also has an inverted "N" shape similar to the isolation enclosure 502 shown in FIGS. 5A and 5B. However, the displacement force generation region in this embodiment includes three sub-regions 566, 567, and 568 that separate the thermal target 545 and the separation zone 564, and the separation zone 564 is further connected to the contiguous zone 562. The displacement force generation region includes a tip portion 566 that also includes a thermal target 545 and a first fluid connector 567 that has the same dimensions as the displacement force generation region / tip portion 566. The displacement force generation region further includes a second constricted fluid connector 568 that connects to the separation region 564, and the width of the fluid connector 568 (dimensions in the x-axis plane when viewed) is the first fluid connector 567 and / Alternatively, it is narrowed as compared with the separation region 564. The narrowing width of the second fluid connector 567 functions to prevent air bubbles generated at the thermal target 545 from coming into contact with microobjects 504 within the separation region 564. In addition, the narrowing width of the second fluid connector 567 may interfere with the shear flow or cavitation force used to displace the microobject 504, or create an abnormal flow that interferes with the shear flow or cavitation force. Avoid the next flow. In addition, the narrowing width of the second fluid connector 567 prevents the microobject 504 from passing from the separation region to the displacement force generation regions (566, 567, and 568).

FIG. 6A shows the circulation culture enclosure 602 and thermal target 622 of the microfluidic device 600 used to generate the Marangoni effect flow 680 according to one embodiment of the present disclosure. The thermal target 622 has an asymmetric teardrop-like shape that creates bubbles 675 with a temperature gradient that produces a circulating Marangoni effect flow 680 when heated using the light source 660. As mentioned above with respect to FIGS. 4C, 4D, 4E, and 4G, a variety of different asymmetric thermal targets can be used to generate the Marangoni effect flow 680.

In the embodiment shown in FIG. 6A, the portion of the thermal target 622 containing the larger surface area is positioned below the portion of the thermal target 622 containing the smaller surface area. Therefore, as a result, the Marangoni effect flow 680, which can be generated by the temperature gradient on the bubble, is directed from the lower part of the bubble 675 to the upper part of the bubble 675 (directed toward the base end opening 634 of the displacement force generation region 614, and the displacement force. (Directed away from the fluid connector 616 in the generation region 614 + 616), in this case producing a counterclockwise circulating Marangoni effect flow 680.

In the embodiment shown in FIG. 6A, the circulation culture enclosure 602 of the microfluidic device 600 has a contiguous zone 610, a culture region 612, and a displacement force generation region 614 including a fluid connector 616. The circulation culture enclosure 602 can be similar to the isolation enclosure, but is configured to circulate the flow through the main channel when actively circulating. The displacement force generation region 614 has a proximal opening 634 to the microfluidic channel 522 and a distal opening 636 from its fluid connector 616 to the culture region 612. The repeating number element is as defined above. When the thermal target 622 is illuminated with light 660, bubbles 675 are nucleated, creating a circulating flow 680 (Marangoni effect flow) that circulates through both the circulating culture enclosure 602 and the channel 522. Circulation flow 680 can be used to mix fluids and / or displace micro-objects (eg, cells) anywhere within the circulation culture enclosure 602 and adjacent channels 522. The velocity of the flow, and thus its displacement force, can be modulated by modulating the power of the illumination, which can require only 1 milliwatt of power to start. In some cases, the circulating flow 680 may have at least a portion of the flow vector in the same direction as the flow of the medium 530 controlled by the medium module 160 and the medium source 178. Circulation flow 680 can be used to flush the medium within channel 522 into culture area 612. Similarly, the circulation flow 680 can be used to displace and carry out microscopic objects within the circulation culture enclosure 602.

In some other embodiments, the isolation enclosure may include other geometries that may include circuits that produce a circulating (Marangoni effect) flow 680. The circulation culture enclosure 602 shown in FIG. 6A includes a circuit incorporating the main channel 522 (referred to herein as the "open loop" circulation culture enclosure 602), while the geometry of the other enclosure is "closed loop". It may include a circular portion of the microfluidic circuit structure within the isolation enclosure that produces the isolation enclosure (ie, a circuit that does not include any portion of the main channel 522).

Depending on the embodiment and the force of the Marangoni effect flow generated by the bubbles, the open-loop circulating culture enclosure and the closed-loop isolation enclosure can have different sizes and shapes. For example, circuits contained within open-loop and closed-loop isolation enclosures may contain different volumes of fluid. Similarly, the length of the circuit can vary depending on the force of the Marangoni effect flow 680 and the type of thermal target 622 used. The circuit may include the entire channel, as described below with respect to FIGS. 8C and 8D.

FIG. 6B shows a "closed loop" isolation enclosure 604 of a microfluidic disk 620 configured to produce a circulating Marangoni effect flow 682. The isolation enclosure 604 has a shape similar to the lowercase letter "b" (ie, a "b" -like shape). In the isolation enclosure shown in FIG. 6B, the separation zone 632 is positioned directly below the connection zone 630, which has a proximal opening 534 to the microfluidic channel 522. The closed loop isolation enclosure 604 has a circular channel, but any type of circuit (eg, a square or polygonal channel) can be used. Isolation enclosure 604 further comprises an asymmetric thermal target 624, which thermal target produces displacement forces with two fluid connections to the separation region 632, eg, two arms of a circular channel from the separation region 630 and to the separation region 630. It is located within region 638. The thermal target 624 can be heated using a light source 662 to produce a bubble 672 with a temperature gradient, and the bubble 672 can generate a Marangoni effect flow 682 in a closed loop circular channel. Since the circular channel does not open to the main channel 522, the Marangoni effect flow 682 can be used to mix the object or fluid medium independently of the fluid medium in the main channel 522.

FIG. 6C shows the isolation enclosure 606 of the microfluidic device 640 configured to generate the Marangoni effect flow 684. The isolation enclosure 606 includes a separation zone 664 located beneath the connection zone 642, which has a proximal opening 534 to channel 530. The isolation enclosure 606 also surrounds a portion of microfluidic circuit material that provides a closed-loop circular channel with two fluid connections from the displacement force generation region 646 to the isolation region 644. The asymmetric thermal target 626 within the displacement force generation region 646 can be heated using the light source 664 to generate bubbles 674 with a temperature gradient that produces the Marangoni effect flow 684.

FIG. 6D shows isolation enclosure 608 of a microfluidic device 670 configured to generate Marangoni effect flows in alternating directions. Similar to the isolation enclosure 606 of the microfluidic device 640, the isolation enclosure 608 has a separation region 654, a displacement force generation region 656 connected to the separation region via two fluid connections (arms of the circulation channel), and a channel 522. It has a connection area 652 with a proximal opening 534 to. The isolation enclosure 608 has two thermal targets 628, 629 configured to generate Marangoni effect flows (not shown) in alternating directions. The alternating directions of the Marangoni effect flow can be used to produce agitating motion to micro-objects or fluid media within the isolation enclosure 608, which is an effect in mixing and removing micro-objects and media. Can function to provide enhancements.

7A-7F show other embodiments of the isolation enclosure useful for optically driven convection and small object displacement. In each of the embodiments shown in FIGS. 7A-7F, the barrier creates a physical separation of the displacement force generating region from the isolation region of the isolation enclosure. The gap between the barrier and the wall of the isolation enclosure, which may be one barrier module or multiple barrier modules, provides a fluid connection between the two regions, but the separation region of the microobjects. It is configured to prevent the passage from the displacement force generation region to the displacement force generation region. Similarly, the gap between the barrier module and the adjacent barrier module provides a fluid connection between the two regions, but is configured to prevent micro-objects from passing from the separation region to the displacement force generation region. NS. In each case, the barrier module can also be configured to avoid damage to the micro-objects from the direct impact of the forces and displacement forces generated by the optically driven convection, making the micro-objects in the separation region more efficient. Channel shear currents, cavitation forces, or bubble forces can also be assisted to remove. Numbering elements with the same number are equivalent.

In FIG. 7A, the isolation enclosure 704 of the microfluidic device 700 opens towards the microfluidic channel 722 configured to include the flow of the fluid medium 706. The microfluidic channel 706 and the wall of the isolation enclosure can be made from the microfluidic circuit material 716. The isolation enclosure 704 has a contiguous zone 714 with a proximal opening 710 to the microfluidic channel 722. The contiguous zone is fluidly connected to a separation zone 712 where the microobject 702 can be placed and / or maintained. The separation region 712 is further connected to the displacement force generation region 718, which includes a thermal target 724 which can be any thermal target described herein. The isolation enclosure 704 also includes one barrier module 726 that forms a boundary between the separation region 714 and the displacement force generation region 718. There are two fluid connections between the separation region 714 and the displacement force generation region 718, which are the gaps 728 between the barrier 728 and the wall of the isolation enclosure 704. Isolation enclosure 704 can be used in the manner of optically driven convection and micro-object displacement. In some embodiments, the thermal target can be illuminated by a light source, which can be a coherent or non-coherent light source and may or may not be structured. In some embodiments, the thermal target is an additional feature within the isolation enclosure 704, a metal, pattern that may be deposited on the cover above the isolation enclosure 704 or on the surface of the base 708. It can be made from a convertible microfluidic circuit material, or a photoinitiated hydrogel polymer. In some embodiments, the thermal target made for this purpose is a sacrificial feature. In another embodiment, the selected discrete region to be illuminated is the selected position on the top surface 708 of the displacement force generation region or the microfluidic circuit material 726 of the wall. Normally, the top surface 708 or microfluidic circuit material 716, when illuminated, behaves as a sacrificial feature and produces heat, but it is also destroyed by the process. The duration of the illumination can determine what kind of displacement force is being generated. In a non-limiting example, short pulses described herein, ranging from about 10 microseconds to about 200 microseconds, can produce cavitation forces for displacement. In a non-limiting example, illumination with a longer duration of about 1000 ms to about 2000 ms can remove one or more microscopic objects within the separation region 712, bubble contact force, bubble flow force. , Bubble meniscus force, or one of the shear flow forces may be provided. The force to remove one or more micro-objects may be sufficient to displace the cell entirely from the isolation region to the microfluidic channel 722, or sufficient to remove the micro-object from the surface of the isolation region 712. However, it is not sufficient to remove cells from the isolation enclosure 704.

FIG. 7B shows another configuration in which the isolation enclosure 730 of the microfluidic device 720 has a plurality of barrier modules 726a that separate the displacement force generation region 718 from the separation region 712. The displacement force generation region 718 is fluidly connected to the separation region 712 via a plurality of fluid connections, a gap 728a.

FIG. 7C represents another variant of the isolation enclosure 732 of the microfluidic device 740 in which the plurality of elongated barrier modules 726b separate the displacement force generation region 718 from the separation region 712. The displacement force generation region 718 is fluidly connected to the separation region 712 via a plurality of fluid connections, a gap 728b.

FIG. 7D is yet another variant with isolation enclosure 734 for the microfluidic device 750. One barrier module 726c has an arc configured to protect micro-objects from direct impact. The displacement force generation region 718 is fluidly connected to the separation region 712 via two fluid connections, a gap 728c.

FIG. 7E is a further variant of the microfluidic device 760 with isolation enclosure 736. One barrier module 726d has a narrow protrusion in the configuration that can help to more efficiently direct the displacement force to displace a small object. The displacement force generation region 718 is fluidly connected to the separation region 712 via two fluid connections, the gap 728d.

FIG. 7F represents another deformation of the isolation enclosure 738 of the microfluidic device 780, in which the plurality of barrier modules 726e generate displacement forces as a circular region surrounding the thermal target 724 and separating the region 718 from the separation region 712. Region 718 is defined. The displacement force generation region 718 is fluidly connected to the separation region 712 via a plurality of fluid connections, a gap 728e.

Isolation enclosures 730, 732, 734, 736, and / or 738 can be made in any similar manner to the construction of isolation enclosures 704, with optical driven convection and / or microobject displacement as a method described for isolation enclosures 704. Can be used in any of the ways.

FIG. 8A shows a microfluidic device comprising a series of isolation enclosures 802, 804, 806 arranged within a channel and having asymmetric thermal targets 840, 842, 844 extending within isolation enclosures 802, 804, 806. The thermal target 842 can be heated using the light source 860 to nucleate bubbles 870 with a temperature gradient, and the temperature gradient interferes with or displaces the micro-object 504 within the isolation enclosure 804 to channel 822. It produces a Marangoni effect flow 880 that can be used to generate a circulating flow within the isolation enclosure 804. The Marangoni effect flow 880 can also be used to introduce a fluid medium from channel 822 into isolation enclosure 804. The thermal targets 840, 842, 844 shown in FIG. 8A are positioned above the isolation enclosures 802, 804, 806 and can be used to introduce the fluid medium into the non-sweep position of the isolation enclosures 802, 804, 806. Although possible, a thermal target configured to produce the Marangoni effect flow 880 can be positioned anywhere in the microfluidic circuit if it is beneficial to introduce the fluid from the swept region to the non-swept region. The velocity, and the resulting force of the circulating flow, can be modulated by increasing or decreasing the power of the illumination, thereby accelerating or decelerating the velocity of the circulating flow.

FIG. 8B shows a microfluidic device 810 with an asymmetric thermal target 846 located at the end of channel 822. If the thermal target 846 is used to generate bubbles 872 with a temperature gradient, the resulting Marangoni effect flow 882 may be used in place of or in combination with channel 830 within the channel to channel 822. You can move the objects inside.

FIG. 8C shows another microfluidic device 812 that includes a main channel 824 and 10 side channels 826a-j extending vertically from the main channel 824. Each of the 10 side channels connects to another side channel to form a microfluidic circuit with a main channel 824. In particular, 826a is connected to 826b, 826c is connected to 826d, 826e is connected to 826f, 826g is connected to 826h, and 826i is connected to 826j to form a circuit. Each of the five microfluidic circuits thus formed contains asymmetric thermal targets 848a-e configured to generate bubbles that give rise to the Marangoni effect flow. In the embodiment shown in FIG. 8C, the main channel 824 has a much lower fluid resistance than the side channels 826a-e. Due to the difference in fluid resistance between the main channel 824 and the side channels 826a to e, the flow of the fluid medium introduced into the main channel 824 does not enter the side channels 826a to e. In other words, the side channels 826a-e are non-sweep regions of the microfluidic device 812 in the absence of a circulating flow guided by optical illumination.

Depending on the embodiment, the ratio of fluid resistance between the main channel 824 and the side channel can vary. In most embodiments, the fluid resistance in the main channel 824 at the point of branching to the side channel 826 is 1/10 to 1/100 of the fluid resistance in the side channel 826. Since fluid resistance is proportional to channel length and inversely proportional to channel width, side channels are typically longer and narrower than the main channel to achieve the optimum ratio of fluid resistance between the main channel and the side channels. .. In some embodiments, the main channel can be 1.5 to 3 times wider than the side channel. For example, the main channel can have a width in the range of 100 μm to 1000 μm and the side channel can have a width in the range of 20 μm to 300 μm.

However, as shown in FIG. 8D, when bubbles 874a and 874b are generated using the asymmetric thermal targets 848a and 848c, the resulting Marangoni effect flow produced by the bubbles 874a and 874b is in the main channel 824. Used in conjunction with the flow, the fluid medium can be selectively introduced from the main channel 824 into the side channels 826a, 826b, 826e, 826f. In this way, the Marangoni effect flow is used to selectively introduce a medium containing specimens, reagents, or micro-objects (eg, beads) into the channel of interest to perform the assay or micro-objects. Can be cultivated.

Kits Kits of optical drive devices and methods for convection and / or small object displacement are provided, the kits being any microfluidic device described herein and described herein in any combination. Includes a microfluidic device, which may include any feature, and a reagent that provides a coated surface. Microfluidic devices include any microfluidic device 100, 200, 230, 250, 280, 290, 500, 550, 560, 600, 620, 640, 670, 700, 720, 720, 750, 760, 780, 808, You can choose from 810, 812, 900, 1000, 1100, 1200, 1300, 1400, 1500. The coated surface reagent can be any reagent described herein for that purpose. Reagents for providing a coated surface may include reagents that provide a covalently bonded surface.

In some embodiments of the kit, one or more fluid media may be provided. In other embodiments, the kit may comprise a photoinitiable hydrogel, which may already be formed as a fluid polymer, or may be a dry powder or lyophilized product. In some embodiments, the kit may further comprise a photoinitiator. The components of the kit may be provided in one or more containers.

Methods of Making Isolation Enclosures with Thermal Targets Thermal targets can be made during a given manufacture of microfluidic devices. The metal target can deposit on the cover of the microfluidic device during the same operation, such as adding a metal contact to the electrical connection. Thermal targets made from microfluidic circuit materials can be included in the mask during soft lithography. Thermal targets placed in this way may include sacrificial targets. The bubble-forming surface topography can be patterned on the microfluidic circuit structure 108 or cover 110 during fabrication, or can be patterned in situ using a light source (not shown). In embodiments where patternable materials are used, structural light sources may be used. The hydrogel thermal target can be installed after the microfluidic device has been made, but before it is used in the methods of the present disclosure.

A method of removing one or more micro-objects and / or mixing a fluid medium Therefore, a method of removing one or more micro-objects (eg, biological micro-objects such as cells) from a surface in a micro-fluid device. Is provided to illuminate a selected discrete region containing or adjacent to one or more micro-objects placed in a fluid medium in the enclosure of a micro-fluid device, the enclosure flowing. Illumination, including microfluidic circuits including regions and substrates, maintaining illumination of selected discrete regions for a first time period sufficient to generate removal forces, one or more microobjects from the surface To remove.

The method may include maintaining one or more micro-objects in a fluid medium in an enclosure for a second time period prior to performing the step of illuminating the selected discrete region. During the maintenance of cells in the fluid medium within the enclosure, some types of cells may attach to one or more inner surfaces of the enclosure of the microfluidic device. Attachment can be a non-specific or specific interaction between the cell and one or more surfaces. Specific interactions can include covalent or non-covalent attachment of surface portions, such as cellular carboxylic acids, to surface oxide moieties, which, when associated, form hydrogen or ester bonds. obtain. Adhesion of one or more cells can be direct or indirect to one or more surfaces. A non-limiting example of direct attachment is the interaction of a portion of one or more cells with an oxide moiety on the surface that has an oxide moiety on the surface. A non-limiting example of indirect attachment of one or more cells to a surface is a portion of the cell (including, but not a limitation, a portion on the surface of the cell) and itself present within the enclosure. It may include interactions with intervening substances or materials that will be associated with, but not limited to, surfaces to which proteins produced by other cells are attached. These are non-limiting examples of possible types of attachment between cells and surfaces on which cells are maintained. Any type of attachment can reduce the portability of one or more cells.

In various embodiments, the enclosure of the microfluidic device may further include at least one isolation enclosure. In some embodiments, the microfluidic device may include multiple isolation enclosures. Each of the plurality of isolation enclosures may have a proximal opening to the flow region. In some embodiments, the flow region may include microfluidic channels.

In some embodiments, the surface on which one or more microobjects are maintained can be the surface of the substrate. The surface of the substrate on which one or more microobjects are maintained can be the surface of the substrate in at least one isolation enclosure.

In various embodiments, the steps of illuminating the selected discrete region are about 1 mm, about 0.9 mm, about 0.7 mm, about 0.5 mm, about 0.3 mm, about 100 μm, about 80 μm, about 60 μm, about. It may include illuminating an area having 40 μm, about 20 μm, about 10 μm, about 5 μm, or any first dimension between them (eg, the width or x-axis dimension of the microfluidic enclosure). The steps of illuminating the selected discrete region are about 1 mm, about 0.9 mm, about 0.7 mm, about 0.5 mm, about 0.3 mm, about 100 μm, about 80 μm, about 60 μm, about 40 μm, about 20 μm, about. It may further include illuminating areas with 10 μm, about 5 μm, or any second dimension between them (eg, height within a microfluidic enclosure or y-axis dimension). The x-axis dimension or the y-axis dimension can be any combination of the above dimensions. Selected discrete regions of illumination are about 200 square μm, about 150 square μm, about 100 square μm, about 80 square μm, about 70 square μm, about 50 square μm, about 25 square μm, about 10 square μm, or It can have an area of any value between them.

Duration of Illumination The step of illuminating can be performed using any of the light sources described herein and can be coherent or non-coherent light. The light can be structural light or non-structural light. For brevity, the following description refers to laser illumination, but the invention is not so limited.

In various embodiments, the step of illuminating the selected discrete region may include illuminating the selected discrete region with a laser. The laser may irradiate with light having a wavelength in the region of about 450 nm to about 800 nm. The laser is about 0.5 amps, about 0.7 amps, about 0.9 amps, about 1.1 amps, about 1.4 amps, about 1.6 amps, about 1.6 amps, about 2.0 amps. , About 2.2 amps, about 2.5 amps, about 2.7 amps, about 3.0 amps, or any value between them.

Laser illumination has incident power in the range of about 1 mW to about 1000 mW, about 100 mW to about 1000 mW, about 100 mW to about 800 mW, about 100 mW to about 600 mW, about 100 mW to about 500 mW, or any value between them. obtain.

In various embodiments, the step of illuminating the selected discrete region with laser illumination can be performed over a time period ranging from about 10 microseconds to about 8000 ms and can be any value between them. In some other embodiments, the step of illuminating the selected discrete region can be performed over a time period ranging from about 100 milliseconds to about 3 minutes.

In various embodiments, the laser illumination is about 50 ms, about 75 ms, about 100 ms, about 150 ms, about 250 ms, about 500 ms, about 750 ms, or about 1000 ms. Can be directed to selected discrete regions over. In various embodiments, the laser illumination is about 50 ms to about 2000 ms, about 50 ms to about 1000 ms, about 50 ms to about 500 ms, about 50 ms to about 300 ms, About 100 ms to about 1000 ms, about 200 ms to about 1000 ms, about 200 ms to about 700 ms, about 300 ms to about 600 ms, or any between any of these ranges. It can be directed to the selected discrete region over a period of time in the range of values of. In other embodiments, the laser illumination is about 1 ms to about 200 ms, about 1 ms to about 150 ms, about 1 ms to about 100 ms, about 1 ms to about 50 ms, About 1 millisecond to about 30 milliseconds, about 25 milliseconds to about 200 milliseconds, about 25 milliseconds to about 100 milliseconds, about 25 milliseconds to about 75 milliseconds, about 50 milliseconds to about 200 milliseconds, It can be directed to selected discrete regions over a time period ranging from about 50 ms to about 125 ms, about 50 ms to about 90 ms, or any value between any of these ranges. obtain. The illumination period selected within one of these ranges may be sufficient to contact the micro-objects and thereby optically drive the formation of bubbles to remove the micro-objects.

In various other embodiments, the laser illumination is about 500 ms to about 3000 ms, about 1000 ms to about 2700 ms, about 1000 ms to about 2500 ms, about 1000 ms to about 2000 ms. Seconds, about 1000 ms to about 1500 ms, about 1300 ms to about 3000 ms, about 1300 ms to about 2700 ms, about 1300 ms to about 2300 ms, about 1300 ms to about 2000 ms Seconds, about 1300 ms to about 1700 ms, about 1500 ms to about 3000 ms, about 1500 ms to about 2600 ms, about 1500 ms to about 2300 ms, about 1500 ms to about 2000 ms Selected over a time period of seconds, from about 1700 ms to about 3000 ms, from about 1700 ms to about 2600 ms, from about 1700 ms to about 2000 ms, or any value between them. Can be directed to discrete regions. Illumination periods selected within one of these ranges may be suitable for the generation of optically driven shear flow or bubble flow contact forces.

In yet another embodiment, the steps of illuminating the selected discrete region are about 10 microseconds to about 200 ms, about 10 microseconds to about 100 ms, about 10 microseconds to about 1 ms, about 10. Microseconds to about 1 millisecond, about 10 microseconds to about 500 microseconds, about 50 microseconds to about 1 millisecond, about 50 microseconds to about 500 milliseconds, about 50 microseconds to about 300 microseconds, about 1 Milliseconds to about 200 milliseconds, about 1 millisecond to about 150 milliseconds, about 1 millisecond to about 100 milliseconds, about 1 millisecond to about 50 milliseconds, about 1 millisecond to about 30 milliseconds, about 25 Milliseconds to about 200 milliseconds, about 25 milliseconds to about 100 milliseconds, about 25 milliseconds to about 75 milliseconds, about 50 milliseconds to about 200 milliseconds, about 50 milliseconds to about 125 milliseconds, about 50 It can run from milliseconds to about 90 milliseconds, or can be any value between these arbitrary ranges. Illumination periods within such an illumination range include tiny objects or generate cavitation forces within discrete selection areas adjacent to tiny objects, thereby removing one or more of the tiny objects. Can be enough. In some embodiments, the duration of illumination can range from about 10 microseconds to about 500 microseconds or from about 10 microseconds to about 100 milliseconds.

In some other embodiments, the steps of illuminating the selected discrete region are about 100 ms to about 3 minutes, about 100 ms to about 2 minutes, about 100 ms to about 1 minute, about 100 ms. Seconds to about 10,000 milliseconds, about 100 milliseconds to about 5,000 milliseconds, about 100 milliseconds to about 1000 milliseconds, about 500 milliseconds to about 3 minutes, about 500 milliseconds to about 1 minute, about It can be performed over 500 ms to about 10,000 ms, about 500 ms to about 3,000 ms, or any value in between. A lighting period within a range selected from one of these ranges may be sufficient to generate a circulating flow (Marangoni effect) that mixes the fluid medium and / or micro-objects. The illumination period of the circular flow can be increased or decreased depending on the power used to illuminate the selected discrete regions.

These ranges are merely exemplary and are not intended to limit this disclosure. While still within the scope of the present disclosure, it is possible to identify and use lighting periods outside the range described for each type of convection or displacement force.

In some embodiments, the step of illuminating a discrete region comprises directing laser illumination to a selected discrete region that includes at least one of one or more microobjects. This can be done anywhere in the enclosure of the microfluidic device. In some embodiments, the illuminated discrete region can be within the isolation enclosure and can be the surface of the substrate within the isolation enclosure. When illuminating at least one micro-object of one or more micro-objects in an isolation enclosure, the selected discrete region is located at the tip of the isolation enclosure's proximal opening to the flow region (eg, isolation). It can be selected to be the bottom or base of the enclosure) or the central location within at least one isolation enclosure).

Laser illumination can directly generate a removal force on at least one of one or more micro-objects. Without being constrained by theory, lighting can, in addition or as an alternative, heat a portion of the fluid medium surrounding one or more micro-objects to remove at least some of the one or more micro-objects. Can generate removal power.

In another embodiment of the method, the illuminated selected discrete region may be adjacent to one or more microobjects. The selected discrete regions are about 1 mm, about 0.9 mm, about 0.7 mm, about 0.5 mm, about 0.3 mm, about 100 μm, about 80 μm, about 60 μm, from one or more microobjects to be removed. It can be located at about 40 μm, about 20 μm, about 10 μm, about 5 μm, or any value between them, apart. Laser illumination adjacent to one or more micro-objects can be performed anywhere within the enclosure of the microfluidic device. In some embodiments, the step of illuminating a selected discrete region adjacent to one or more micro-objects can be performed on a substrate, on a wall microfluidic circuit material, or on a thermal target and is a thermal target. Can be any thermal target described herein, which can also be a sacrificial feature. In some embodiments, if one or more micro-objects are maintained within the isolation enclosure, the step of illuminating the substrate is on the substrate near the proximal opening of at least one isolation enclosure to the flow region. It can be performed in a selected discrete region. In other embodiments, if one or more micro-objects are maintained within at least one isolation enclosure, the step of illuminating the selected discrete region is the selection of at least one isolation enclosure microfluidic circuit material. It may include illuminating a discrete area. In yet another embodiment, if one or more micro-objects are maintained within at least one isolation enclosure, the step of illuminating the selected discrete region is a sacrificial feature placed within at least one isolation enclosure. May include illuminating.

Sacrificial features can be made of any suitable material capable of absorbing energy from laser illumination, with metal pads or walls of microfluidic circuit materials (eg, walls of isolation enclosures and flow regions (eg, microfluidic channels)). The same or similar material) can be included. In some embodiments, the sacrificial feature may or may not include an additional coating or covalently modified surface layer of the substrate or any other that may be included within the microfluidic device. May include material.

Illumination (including, but not limited to, laser illumination, for example) can directly generate a removal force on at least one of one or more micro-objects. Without being constrained by theory, lighting, as an addition or alternative, heats a portion of the fluid medium around one or more micro-objects, producing a cavitation-removing force capable of removing one or more micro-objects. obtain.

Laser illumination can directly generate a removal force on at least one of one or more micro-objects. Unconstrained by theory, the step of illuminating a selection area adjacent to one or more micro-objects, additionally or as an alternative, heats a first portion of the fluid medium and surrounds one or more micro-objects. Persistent bubbles that displace a second portion of the fluid medium can be created, thereby removing one or more microscopic objects. The step of removing the second portion of the fluid medium may further include creating a circulating fluid flow of the fluid medium during the first illumination period. In another embodiment, the method heats a first portion of the fluid medium and creates one or more bubbles, thereby creating a shear flow of the fluid medium towards one or more microobjects. It may further include producing. In yet another embodiment, the method is to heat a first portion of the fluid medium and to generate a plurality of bubbles configured to flow towards one or more micro-objects. Alternatively, it may further include contacting the plurality of microobjects with the meniscus of at least one bubble of the plurality of bubbles.

Laser illumination can be directed to any of the locations mentioned above. Alternatively, if one or more micro-objects are maintained within the isolation enclosure within the enclosure, the selected discretes may include at least a portion of the wall forming the tip of the isolation enclosure, the wall. Positioned opposite to the proximal opening to the flow region. Lighting at the base of the isolation enclosure can avoid damage to one or more micro-objects.

Alternatively, if one or more micro-objects are placed within the isolation enclosure within the enclosure, the selected discrete region may be located in the displacement force generation region of the isolation enclosure. In some embodiments, one or more microobjects may be placed within the isolation region of the isolation enclosure, and the displacement force generation region is fluidly connected to the isolation region.

In various embodiments of the method of removing one or more micro-objects in a microfluidic device, the method may further include the step of ejecting one or more micro-objects from at least one isolation enclosure. The step of removing one or more micro-objects from at least one isolation enclosure can include moving one or more micro-objects using dielectrophoretic forces.

In various embodiments of the method of removing one or more micro-objects in a microfluidic device, the method may further include the step of ejecting one or more micro-objects from the flow region of the enclosure of the microfluidic device. .. The step of removing one or more microobjects from the flow region may include the use of gravity, fluid flow, dielectrophoretic forces, or any combination thereof.

In certain embodiments, the present disclosure further provides a machine-readable storage device that stores non-transient machine-readable instructions that perform the above method. Machine-readable instructions can also control the imaging device used to acquire the image.

In another aspect, a method of mixing a fluid medium and / or microobjects contained in the fluid medium within the enclosure of a microfluidic device is provided, the method being a microfluidic circuit comprising at least one fluid medium and / or microobjects. A step of focusing the light source on a thermal target located on the surface of the inner enclosure, thereby heating and focusing the first portion of at least one fluid medium, and at least one in the microfluidic circuit. A step of inducing a circulating flow of one fluid medium, thereby including a step of mixing and guiding the fluid medium and / or microobjects arranged therein. In some embodiments of the method, the thermal target is located within the first microfluidic channel and the first microfluidic channel is configured to branch into a second fluid channel at the first position. , Also configured to recombine to the second fluid channel in the second position, the thermal target is placed on the surface between them.

Experimental system and microfluidic device: manufactured by Berkeley Lights, Inc. The system included at least a flow controller, temperature controller, fluid medium conditioning and pump components, light source for photoactivated DEP configuration, laser, on-board stage of Berkeley Lights, Inc. OptoFluidic ™ microfluidic device, and camera. .. The Berkeley Lights, Inc. OptoFluidic ™ microfluidic device included a NanoPen ™ chamber with a volume of ^{approximately 7 × 10 5 cubic μm.}

Material: The cells were OKT3 cells (ATCC® Catalog No. CRL-8001 ™), a mouse myeloma hybridoma cell line, obtained from ATCC, unless otherwise stated. The cells were provided as a suspended cell line. Using 5% carbon dioxide in the air as the gas environment, about 1 × 10 ⁵ to about 2 × 10 ⁵ viable cells per mL were seeded and cultured at 37 ° C. to maintain the culture. Cells were divided every 2-3 days. The number and viability of OKT3 cells were counted and the cell density was adjusted to ^{5 × 10 5 / mL for loading into microfluidic devices.}

Culture Medium: 500 mL of Iskov modified Darveco medium (ATCC® Catalog No. 30-2005), 200 mL of fetal bovine serum (ATCC® Catalog No. 30-2020), and penicillin-streptomycin (Life Technologies®).
Catalog number 15140-122) 1 mL was mixed to prepare a medium. The finished medium was filtered through a 0.22 μm filter and stored at 4 ° C., avoiding light until use.

Priming procedure: 250 μL of 100% carbon dioxide was flowed in at a flow rate of 12 μL / sec. After this, 0.1% Pluronic® F27 (Life Technologies®)
250 μL of PBS containing Catalog No. P6866) was flowed in at a flow rate of 12 μL / sec. The final step of priming included influx of 250 μL PBS at a flow rate of 12 L / sec. After that, the introduction of the medium continued.

Perfusion method (during cell culture on the chip: The perfusion method was one of the following two methods.
Perfusion at 1.0.01 μL / sec for 2 hours, perfusion at 2 μL / sec for 64 seconds, and repeat.
2. Perfusion at 0.02 μL / sec for 100 seconds, stop flow for 500 seconds, perfuse at 2 μL / sec for 64 seconds, and repeat.

Optical system: In Examples 1 and 2, the optical system included a 785 nm laser Olympus microscope Prosilica camera and an epi-illumination optical train with a dedicated collimating optics for the laser.

Example 1. Optical Illumination of Metal Thermal Targets FIGS. 9A-9D show the use of thermal targets to generate bubbles used to eject cells from the isolation enclosure. The isolation enclosure 902 of the microfluidic device 900 shown in FIGS. 9A-9C has an inverted "N" -like geometry similar to the isolation enclosure shown in FIG. 5A, with a connection region 906 and a human. Displacement force including a separation region 908 in which hybridoma cells 904 are cultured and a three-part fluid connector 909 having a proximal constriction segment 912 connecting to the separation region and a tip constriction segment 914 connecting to the reservoir region 913 of the displacement force generation region 910. Includes a generation region 910 (marked in FIG. 9C). The proximal constriction segment 912 of the displacement force generating region 910 has a width smaller than the diameter of the cells, preventing any cells from moving from the separation region 908 to the displacement force generating region 910, in particular the optical illumination is focused. Cells are isolated from the reservoir region 913, which has the highest heating intensity. The displacement force generation region further includes a thermal target 916, which is a continuous metallic shape formed of gold (Au) deposited on the inner surface of the cover of the microfluidic device, in the reservoir region 913. FIG. 9A shows an isolation enclosure with multiple cells prior to optical illumination and after culturing in the isolation region for 3 days.

Thermal targets were heated for 5-10 seconds using a 785 nm laser with currents in the range of 0.8 amps to 1.0 amps. FIG. 9B is a photograph taken at a time during the illumination period, in which bubbles (not shown) are formed in the thermal target 916, displace the cells from the isolation region 908, and displace the cells 904 in the connecting region 906 and It was carried out to the microfluidic channel 922 near the isolation enclosure 902. FIG. 9C shows the quarantine enclosure at the time after the cells were ejected. The exported cells 904 were transferred to a standard well plate and seeded individually. After culturing in a well plate for 3 days, cells 904 demonstrated viability by growing into a larger cell population (Fig. 9D).

Example 2. Optical Illumination of Sacrificial Features Producing Bubble Flow Figures 10A and 10B show the export of human hybridoma cells from isolation enclosure 1002 with connection area 1006 and separation area 1008. In this example, the isolation enclosure did not have discrete or separate displacement force generating regions.

Cells were cultured in isolation enclosure 1002 of microfluidic device 1000 for 3 days (not shown). A laser with a 1.4 amp current (power was 90 milliwatts) was applied to the inner surface (thermal target) of a microfluidic circuit that was a cover containing a dielectric transfer substrate and indium tin oxide (“ITO”) for 5 seconds. Carrying out was carried out by focusing for 10 seconds. The particular location illuminated was the selected discrete region 1020 of the inner surface at the base of the isolation enclosure 1002, as opposed to the opening of the isolation enclosure to the microfluidic channel 1022. The thermal target was a substrate that absorbed optical illumination and converted the illumination into thermal energy, thereby heating the surrounding fluid medium. This process destroyed part of the substrate that served as a sacrificial feature. The fluid medium was heated sufficiently to nucleate the flow of bubbles. FIG. 10A shows the time point during the illumination period when the bubbles 1024 were formed in the lower part of the isolation enclosure 1002 by focusing the light on the substrate. Cell 1004 was moved from isolation zone 1008 to contiguous zone 1006 isolation enclosure. FIG. 10B shows the isolation enclosure 1002 at a later time point in which the volume of flow of bubbles 1024 grew and most of the cells 1004 were expelled from the enclosure 1002 into the microfluidic channel 1022. The carried-out cells 1004 were transferred to a well plate and individually seeded for further culturing. FIG. 10C shows the well of one of the well plates after 3 days, showing that the individually seeded cells were alive and proliferated.

Optical system of Examples 3 and 4 The optical train was modified to incorporate the Chroma ZT745spxrxt-UF1 dichroic filter (Chroma, Below Falls, Vermont), ZET785nf (Chroma) emission filter, and 4X Nikon objective, which are 785nm lasers. ..

Protocols of Examples 3 and 4. A 785 nm laser is focused on the inner surface of the isolation enclosure (dielectrophoresis substrate) and the cover above the enclosure to generate 90 milliwatts (mW) of power for about 1 second, heating the fluid medium to create one or more bubbles. Generated. Cells were removed from each separation region by the bubble contact force and shear flow generated by the bubbles. After removal, OET forces were used to deliver and position the removed individual cells into an adjacent enclosure. After 24 hours in culture, freshly repositioned cells were observed to identify their effects on cell viability and proliferation.

Example 3. Optically driven displacement and viability of OKT3 cells FIGS. 11A-11C show the experimental results before and after the optically driven displacement and repositioning of OKT3 cells. FIG. 11A shows a series of isolation enclosures for the microfluidic device 1100 prior to export of cells 1104 from isolation enclosure 1102. Optical illumination was directed at the sacrificial feature 1120, which is the surface of the isolation enclosure 1102, which acts as a thermal target. As a result, cells 1104 were removed. One cell was positioned in an adjacent isolation enclosure. FIG. 11B shows individual cells 1104b, 1104c, 1104d, and 1104e that were removed and positioned within the newly occupied enclosure after being displaced and repositioned using OET. The number of cells 1104a in the originally occupied isolation enclosure 1102 was reduced and the cells were removed but may remain in the isolation enclosure 1103. FIG. 11C shows the same isolation enclosure 20 after a few hours. As shown in FIG. 12C, cells 1104b, 1104c, 1104d, and 1104e continued to divide and proliferate after removal, unloading, and repositioning. In addition, residual cells in the originally occupied isolation enclosure 1102 also proliferated. These results indicate that the processes of optical illumination, heating, removal, and repositioning had no detectable effect on cell viability in this experiment. As shown in FIG. 12C, the number of cells 1104c increased from 2 to 4, the number of cells 1104d increased from 1 to 4, and the number of cells 1104e increased from 1 to 2.

Example 4. Optically Driven Displacement of JIMT-1 Cells and Consequent Viability Medium: Serum-Free Medium with Medium Conditioning Additive B-27® Supplement (2% v / v) (Thermo Fisher Scientific Catalog No. 12045-096) ).

12A-12C show the experimental results before and after the optical drive displacement and repositioning of JIMT-1 cells (commercially available from AddexBio Catalog No. C6000605), which are adherent human breast cancer cell lines. FIG. 12A shows a series of isolation enclosures for the microfluidic device 1200 prior to displacement of cells 1204 from isolation enclosure 1202. Cell 1204 was removed by directing optical illumination to the sacrificial feature 1220 (to the thermal target), which is the surface of the isolation enclosure 1202. FIG. 14B shows individual cells 1204a removed and newly repositioned in an empty quarantine enclosure adjacent to the originally occupied quarantine enclosure 1202. FIG. 14C shows the time point 20 hours after displacement and repositioning, showing that cells 1204a were alive and doubled from one to two within the 20 hour culture period.

Example 5. Introduction of Circulating (Marangoni Effect) Flow FIGS. 13A-13C show experimental results showing a circulating Marangoni effect flow using a circulating culture enclosure geometry. FIG. 13A shows some microscopic points at the first time point when the laser was directed at the thermal target 1320 to create bubbles 1330 in the circulating culture enclosure 1302 and adjacent channel 1322 of the microfluidic device 1300 to produce a Marangoni effect flow. Object 1305 (polystyrene beads with a diameter of 6 μm) is shown. FIG. 13B shows the micro-object 1305 circulated counterclockwise through the culture enclosure 1302 by the Marangoni effect flow (white arrows indicate the flow direction) at the second time point (after the bubbles 1330a broke away from the nucleated portion). ) Shows the same circulation culture enclosure 1302 with microobjects 1305. FIG. 13C shows a third time point in which laser illumination was still present. The microobject 1305 is pushed past the thermal target 1320 into the channel 1322 and circulates back to the second side of the circulation culture enclosure 1302. In the experiments shown in FIGS. 13A-13C, bubbles were nucleated by focusing a 785 laser on a gold thermal target deposited on the inner surface of the cover of a microfluidic device. In particular, the laser was used to generate light spots with a diameter of 40 μm and had 90 mW corresponding to ^{1.4 kW / cm 2.}

Example 6. Optically Driven Displacement of OKT3 Cells in Shorter Illumination Periods OKT3 mouse hybridoma cells were cultured in a fluid medium within the isolation enclosure of the microfluidic device 1400 shown. FIG. 14A shows a colony of cells 1440 maintained within the central isolation enclosure 1402. The cell population is highlighted within a white oval and laser illumination has not yet been introduced. FIG. 14B shows selected discrete regions on the surface of isolation enclosure 1302 containing some of the cells, with the laser using 1.4 amperes for a duration ranging from about 50 ms to about 1000 ms. The same group of cells 1440 (inside the white oval) within the central isolation enclosure 1402 in the figure is shown while being directed at 1420 (the sacrificial feature thermal target). FIG. 14C showed that the cavitation and decay of nucleated bubbles from the heat introduced by laser illumination completely removed the group of cells 1404b from the isolation enclosure 1402 and displaced it into the microfluidic channel 1422. .. The rest of the cells 1404a were displaced somewhat but were not removed from the isolation enclosure 1402.

Example 7. Optically Driven Displacement of OKT3 Cells at Longer Illumination Period OKT3 mouse hybridoma cells 1504 were maintained in a fluid medium within the isolation enclosure of the microfluidic device 1500, and all pre-laser illumination cells 1504 are shown in FIG. 15A, in FIG. 15A. The white oval points to the cell colonies to be removed. Laser illumination is shown in FIG. 15B. The laser power was 1.4 amps and the duration of the laser pulse was about 2000 ms. The white oval surrounds the cells 1504 to be removed, and the discrete region of illumination 1520 is below the isolation enclosure 1502, specifically directed to the microfluidic circuit material that forms the walls of the isolation enclosure. The targeted area acted as a sacrificial feature (eg, a thermal target). FIG. 15C shows a time point near the end of 2000 ms laser illumination, at which point cells 1504 (inside the white oval) were removed and pushed by air bubbles under the population of cells 1504 (invisible) to flow. Displaced towards the proximal opening of the isolation enclosure to the region (eg, microfluidic channel). The blackening seen at the base of the isolation enclosure indicates some destruction of the substrate material within the isolation enclosure 1502 and the walls of the isolation enclosure (see also post-illumination sacrifice feature 1524 in FIG. 15D). FIG. 15D shows the time point after the completion of optical illumination, at which point an optically actuated dielectrophoretic force was applied, where the removed cells 1504 continued to move further away from the isolation enclosure 1504. In FIG. 15D, white bars 1530, 1532, 1534, 1536 are light (OET) patterns displayed on the surface of the substrate, wherein the substrate contains a dielectrophoretic configuration. The light pattern bars 1530, 1532, 1534, and 1536 moved toward the opening of the isolation enclosure to the microfluidic channel 1522, so that the cells captured and excluded by the dielectrophoretic force generated by each light bar pattern Moved towards the opening of the isolation enclosure (see cells 1504b and 1504c in the white oval). The cells 1504c were dispelled by the optical pattern bar 1530, completely removed from the isolation enclosure, and relocated to the flow region (eg, microfluidic channel 1522). FIG. 15E shows a later point in time when the optically actuated dielectrophoretic light patterns 1530, 1532, 1534, 1536 completed a series of cell capture, exclusion, and removal from the isolation enclosure and returned to the fluid flow flow region. show. Cells that were removed by the laser pulse method and further removed from the isolation enclosure into the flow region were removed from the microfluidic device. Cells 1504d (inside the white oval for enhancement) still remained in the isolation enclosure, but were clearly removed from their original position within the isolation enclosure 1502 prior to laser illumination (compared to FIG. 15A). .. An additional sequence of selection and migration by optically working dielectrophoresis can carry the remaining cells out of the quarantine enclosure.

Description of Embodiment 1. A microfluidic device that includes an enclosure that further includes a flow region and an isolation enclosure, the isolation enclosure including a connection region, a separation region, and a displacement force generating region, the connection region being a proximal opening to the flow region and separation. A microfluidic device comprising a tip opening to a region, a separation region comprising at least one fluid connection to a displacement force generating region, and a displacement force generating region further comprising a thermal target.

2. The microfluidic device of claim 1, wherein at least one fluid connection between the separation region and the displacement force generation region comprises a cross-sectional dimension configured to prevent microobjects from passing from the separation region to the displacement force generation region. ..

3. 3. At least one fluid connection between the separation region and the displacement force generation region is configured to prevent the fluid from flowing out of the displacement force generation region in the absence of any internally generated force, except for the force due to diffusion. The microfluidic device according to embodiment 1 or 2, comprising cross-sectional dimensions.

4. At least one fluid connection between the separation region and the displacement force generation region includes one or more barrier modules, which do not allow microscopic objects to pass from the separation region to the displacement force generation region. The microfluidic device according to any one of embodiments 1 to 3, wherein the microfluidic device is configured to be such.

5. The microfluidic device according to any one of embodiments 1 to 5, wherein the displacement force generating region further includes an opening to a flow region.

6. The microfluidic device according to any one of embodiments 1 to 5, wherein the displacement force generating region has two or more fluid connections to the separation region.

7. The microfluidic device according to embodiment 6, wherein the isolation enclosure comprises a circulation channel.

8. The microfluidic device according to embodiment 7, wherein the circulation flow path includes a constricted portion.

9. The microfluidic device according to any one of embodiments 6-8, further comprising a second thermal target configured to generate a second circulating flow of the fluid medium when optically illuminated.

10. The microfluidic device according to embodiment 9, wherein the first thermal target and the second thermal target are directed to provide a first circulation flow and a second circulation flow of the fluid medium in the opposite direction.

11. The microfluidic device according to any one of embodiments 1 to 5, wherein the displacement force generating region comprises one opening, the one opening being a fluid connection to the separation region.

12. The microfluidic device according to any one of embodiments 1 to 11, wherein the fluid connection in the displacement force generating region comprises a fluid connector that includes at least one bend.

13. 12. The microfluidic device of embodiment 12, wherein at least one bend in the fluid connector comprises a turn of about 60 degrees to about 180 degrees.

14. The microfluidic device according to embodiment 12 or 13, wherein the fluid connector in the displacement force generating region comprises at least two bends.

15. The microfluidic device according to embodiment 14, wherein each of at least two bends of the fluid connector comprises a turn of about 60 degrees to about 180 degrees.

16. The microfluidic device according to any one of embodiments 12 to 15, wherein the width of the fluid connector is the same as the width of the separation region and / or the displacement force generation region.

17. The microfluidic device according to any one of embodiments 12 to 15, wherein the fluid connector comprises a cross-sectional dimension configured to prevent microobjects from passing from a separation region to a displacement force generation region.

18. The microfluidic device according to any one of embodiments 1-17, wherein the enclosure of the microfluidic device further comprises a cover that partially defines the isolation enclosure and the thermal target is located on the cover.

19. The microfluidic device of embodiment 18, wherein the thermal target is located on the inner surface of a cover facing the enclosure.

20. The microfluidic according to any one of embodiments 1-17, wherein the enclosure of the microfluidic device further comprises a microfluidic circuit structure that partially defines the isolation enclosure and the thermal target is located in the microfluidic circuit structure. device.

21. The microfluidic device according to any one of embodiments 1-17, wherein the enclosure of the microfluidic device further comprises a base that partially defines the isolation enclosure and the thermal target is located on the inner surface of the base.

22. The microfluidic device according to any one of embodiments 1 to 21, wherein the thermal target comprises a metal.

23. The microfluidic device according to any one of embodiments 1 to 22, wherein the thermal target has a continuous shape.

24. The microfluidic device according to any one of embodiments 1 to 22, wherein the thermal target has a discontinuous shape.

25. The microfluidic device according to any one of embodiments 1-22 or 24, wherein the thermal target comprises a plurality of microstructures.

26. The microfluidic device according to any one of embodiments 1-21 or 23-25, wherein the thermal target is a sacrificial feature.

27. The microfluidic device according to any one of embodiments 1-26, wherein the thermal target or displacement force generating region is configured to limit the expansion of bubbles formed in one main direction.

28. The microfluidic device according to any one of embodiments 1-27, wherein the thermal target is positioned at a portion of the displacement force generating region of the tip to at least one fluid connection to the separation region.

29. 28. The microfluidic device of embodiment 28, wherein the displacement force generating region has a width of about 20 μm to about 100 μm.

30. The microfluidic device according to any one of embodiments 1-29, wherein the enclosure further comprises a dielectrophoretic configuration.

31. 30. The microfluidic device of embodiment 30, wherein the dielectrophoresis configuration is optically actuated.

32. The microfluidic device according to any one of embodiments 1-31, wherein the isolation enclosure comprises at least one surface that is a covering surface.

33. The microfluidic device according to embodiment 32, wherein the coated surface is a covalent bond surface.

34. A microfluidic device that includes an enclosure, the enclosure being a microfluidic circuit configured to include a fluid medium, the microfluidic circuit being configured to correspond to at least one circulating flow of the fluid medium. The microfluidic circuit and the first thermal target located on the surface of the enclosure in the microfluidic circuit, the first thermal target, when optically illuminated, is the first circulating flow of the fluid medium. A microfluidic device, including a first thermal target, which is configured to produce.

35. The microfluidic device according to embodiment 34, wherein the thermal target has a continuous shape.

36. The microfluidic device according to embodiment 34, wherein the thermal target has a pattern of shape.

37. The thermal target comprises a non-uniform thickness on the surface of the enclosure, and the non-uniform thickness is configured to provide different heating of the fluid medium by the thermal target when optically illuminated. The microfluidic device according to any one of embodiments 34 to 36.

38. The microfluidic device according to embodiment 36 or 37, wherein the pattern of shapes is configured to provide different heating of the fluid medium by a thermal target when optically illuminated.

39. The microfluidic device according to any one of embodiments 34-38, wherein the thermal target comprises a metal.

40. The microfluidic device according to any one of embodiments 34-39, wherein the thermal target comprises a plurality of microstructures.

41. 40. The microfluidic device of embodiment 40, wherein the microstructures include a density-increasing pattern of microstructures configured to provide different heating of the fluid medium by a thermal target when optically illuminated.

42. The enclosure of a microfluidic device further comprises a microfluidic channel and an isolation enclosure, further comprising the isolation enclosure adjacent to the microfluidic channel and opening towards the microfluidic channel, according to any one of embodiments 34-41. Microfluidic device.

43. 42. The microfluidic device of embodiment 42, wherein the circulation channel comprises a portion of the channel and at least a portion of the isolation enclosure.

44. 42. The microfluidic device of embodiment 42, wherein the isolation enclosure comprises a circulation channel.

45. The microfluidic device according to any one of embodiments 34 to 44, wherein the circulation channel comprises a constriction.

46. The microfluidic device according to any one of embodiments 34-45, further comprising a second thermal target configured to generate a second circulating flow of the fluid medium when optically illuminated.

47. The microfluidic device according to embodiment 46, wherein the first thermal target and the second thermal target are directed to provide a first circulation flow and a second circulation flow of the fluid medium in the opposite direction.

48. The microfluidic device according to embodiment 42, wherein the thermal target is located on a surface within the microfluidic channel.

49. The enclosure of the microfluidic device further comprises two or more microfluidic channels so that the first microfluidic channel opens from the second microfluidic channel in the first position along the second microfluidic channel. Configured and reconnected to the second microfluidic channel in the second position, thereby further configured to form a microfluidic circuit, the thermal target is located on the surface within the first microfluidic channel. , The microfluidic device according to any one of embodiments 34 to 41.

50. The microfluidic device according to embodiment 49, wherein at least one isolation enclosure opens in a first microfluidic channel.

51. The microfluidic device according to embodiment 49 or 50, wherein the fluid resistance of the first channel is about 10 to about 100 times the fluid resistance of the second channel.

52. The microfluidic device according to any one of embodiments 49-51, wherein the second microfluidic channel comprises a width of about 1.5 to about 3 times the width of the first microfluidic channel.

53. The microfluidic device according to embodiment 52, wherein the width of the second microfluidic channel is from about 100 μm to about 1000 μm.

54. The microfluidic device according to any one of embodiments 49-53, wherein the width of the first microfluidic channel is from about 20 μm to about 300 μm.

55. A microfluidic device that includes an enclosure, the enclosure containing a microfluidic channel and an isolation enclosure, the isolation enclosure adjacent to the microfluidic channel, opening in the microfluidic channel, and a thermal target opening to the isolation enclosure. A microfluidic device that is placed in a channel adjacent to the and is further configured to direct the flow of the fluid medium towards the isolation enclosure when optically illuminated.

56. The microfluidic device according to embodiment 55, wherein the thermal target is located on a surface within the microfluidic channel.

57. The microfluidic device according to embodiment 55 or 56, wherein the thermal target has a continuous shape.

58. The microfluidic device according to embodiment 55 or 56, wherein the target heat has a pattern of shape.

59. The thermal target comprises a non-uniform thickness on the surface of the enclosure, and the non-uniform thickness is configured to provide different heating of the fluid medium by the thermal target when optically illuminated. The microfluidic device according to any one of embodiments 55-58.

60. 28. The microfluidic device of embodiment 58 or 59, wherein the pattern of shapes is configured to provide different heating of the fluid medium by a thermal target when optically illuminated.

61. The microfluidic device according to any one of embodiments 55-60, wherein the thermal target comprises a metal.

62. The microfluidic device according to any one of embodiments 55-60, wherein the thermal target comprises a plurality of microstructures.

63. 62. The microfluidic device of embodiment 62, wherein the microstructures include a density-increasing pattern of microstructures configured to provide different heating of the fluid medium by a thermal target when optically illuminated.

64. Microobjects comprising the microfluidic device according to any one of embodiments 1-33 and one or more reagents configured to provide at least one covering surface within the enclosure of the microfluidic device. Kit to incubate.

65. The kit according to embodiment 64, further comprising at least one fluid medium.

66. A method of removing one or more micro-objects within a microfluidic device, comprising or adjacent to one or more micro-objects placed in a fluid medium in the enclosure of the microfluidic device. A step of illuminating a discrete region, the enclosure includes a microfluidic circuit including a flow region and a substrate, a step of illuminating and a first time period sufficient to generate a removal force to illuminate the selected discrete region. A method comprising the steps of removing and maintaining one or more microobjects from a surface.

67. The method of embodiment 66, wherein the selected discrete region has an area of approximately 100 square μm.

68. The method of embodiment 66, wherein the selected discrete region has an area of approximately 25 square μm.

69. The method of embodiment 66, wherein the illuminating step comprises illuminating a discrete region selected by a laser.

70. The method of any one of embodiments 66-69, wherein the one or more microobjects are placed on the surface of the substrate.

71. Any one of embodiments 66-70, further comprising the step of maintaining one or more microobjects in the fluid medium in the enclosure for a second time period prior to performing the step of illuminating the selected discrete region. The method described in one.

72. The method of any one of embodiments 66-71, wherein the enclosure of the microfluidic device comprises at least one isolation enclosure.

73. The method of embodiment 72, wherein one or more micro-objects are placed and / or maintained on the surface of a substrate in at least one isolation enclosure.

74. The method of any one of embodiments 66-73, wherein the step of illuminating the selected discrete region comprises illuminating with illumination having an incident power in the range of about 1 mW to about 1000 mW.

75. The method of embodiment 74, wherein the first time period ranges from about 10 microseconds to about 3000 milliseconds or from about 100 milliseconds to about 3 minutes.

76. The method of any one of embodiments 66-75, wherein the illuminating step comprises illuminating at least one of one or more microobjects.

77. The method of embodiment 76, wherein the first time period ranges from about 10 microseconds to about 200 milliseconds, thereby generating a cavitation force that removes one or more microobjects.

78.1 If one or more micro-objects are placed and / or maintained within at least one isolation enclosure within the enclosure, the selected discrete region will be to the proximal opening of the isolation enclosure to the flow region. The method according to embodiment 76 or 77, which is located at the tip or at the center of at least one isolation enclosure.

79. The method of any one of embodiments 66-78, wherein the selected discrete region is a selected point adjacent to one or more micro-objects.

80. 29. The method of embodiment 79, wherein the step of illuminating the selected discrete region comprises directing the illumination to a substrate, a wall microfluidic circuit material, or a thermal target.

81. 80. The method of embodiment 80, wherein the thermal target comprises a metal deposit, a pattern of metal deposits, or microstructures patterned on the surface.

82. 80 or 81. The method of embodiment 80 or 81, wherein the thermal target comprises sacrificial features.

83.1 One or more microobjects are placed within at least one isolation enclosure within the enclosure, and the selected discrete regions are located near the proximal opening of at least one isolation enclosure to the flow region. The method according to any one of embodiments 79 to 82.

84. When one or more micro-objects are placed within at least one isolation enclosure within the enclosure, the selected discrete regions are selected microfluidic circuit materials that help define at least one isolation enclosure. The method according to any one of embodiments 79-82, comprising points.

85.1 If one or more micro-objects are maintained within at least one isolation enclosure within the enclosure, the selected discrete region comprises a sacrificial feature that is located within at least one isolation enclosure. The method according to any one of 79 to 82.

86. The method of embodiment 85, wherein the sacrificial feature comprises a microfluidic circuit material.

87. The method according to any one of embodiments 79-86, wherein the first time period is in the range of 10 microseconds to 200 milliseconds.

88. The step of illuminating the selected discrete region further comprises heating a first portion of the fluid medium placed within or adjacent to the selected discrete region, thereby generating a cavitation force. The method according to any one of forms 79 to 87.

89. The method is to heat a first portion of the fluid medium and generate persistent bubbles, thereby displace the second portion of the fluid medium surrounding one or more microscopic objects. The method according to any one of embodiments 79-87, further comprising generating persistent bubbles.

90. The method of embodiment 89, wherein the step of displacing the second portion of the fluid medium further comprises generating a circulating fluid flow of the fluid medium during the first period of illumination.

91. The method is to heat a first portion of the fluid medium and generate one or more bubbles, thereby creating a shear flow of the fluid medium towards one or more microobjects. The method according to any one of embodiments 79-87, further comprising generating air bubbles.

92. The method is to heat a first portion of the fluid medium, generate multiple bubbles configured to flow towards one or more microobjects, and at least one of the multiple bubbles. The method according to any one of embodiments 79-87, further comprising contacting one or more microobjects with a meniscus.

93. The method according to any one of embodiments 89-92, wherein the first time period ranges from about 100 milliseconds to about 3000 minutes.

94. The method of embodiment 91 or 92, wherein the first period is in the range of about 1000 ms to about 2000 ms.

95. If one or more micro-objects are maintained within the isolation enclosure within the enclosure, the selected discrete region comprises at least a portion of the wall forming the tip of the isolation enclosure, the wall flowing. The method according to any one of embodiments 79-94, which is positioned opposite to the proximal opening to the region.

96. If one or more micro-objects are placed within the isolation enclosure within the enclosure, the selected discrete region is located within the displacement force generation region of the isolation enclosure, any one of embodiments 79-94. The method described in one.

97. The method of embodiment 96, wherein the one or more microobjects are placed within a separation region of the isolation enclosure, and the displacement force generation region is fluidly connected to the separation region.

98. The method of any one of embodiments 66-93, further comprising the step of removing the one or more microobjects from at least one isolation enclosure placed within the enclosure.

99. The method of embodiment 98, wherein the step of removing one or more micro-objects from at least one isolation enclosure comprises moving one or more micro-objects using dielectrophoretic forces.

10.0. The method of any one of embodiments 66-99, further comprising the step of removing one or more microobjects from the flow region of the enclosure of the microfluidic device.

101. The method of embodiment 100, wherein the step of removing one or more microobjects from the flow region comprises using fluid flow or dielectrophoretic forces.

102. A method of mixing a fluid medium and / or micro-objects contained in the fluid medium within the enclosure of a micro-fluid device, located on the surface of the enclosure in a micro-fluid circuit containing at least one fluid medium and / or micro-object. Focusing the light source on a thermal target, thereby heating and focusing the first portion of at least one fluid medium and inducing a circulating flow of at least one fluid medium in the microfluidic circuit. A method comprising: mixing and guiding a fluid medium and / or microobjects placed within the fluid medium thereby.

103. The thermal target is configured to branch into a second fluid channel at the first position and is also configured to recombine to the second fluid channel at the second position. 10. The method of embodiment 102, wherein the thermal target is placed on a surface between them.

Although specific embodiments and applications of the present disclosure have been described herein, these embodiments and applications are merely exemplary and many modifications are possible.

Claims (27)

A method of removing one or more microscopic objects within a microfluidic device.
The microfluidic device comprises an enclosure, the enclosure comprising a base and a microfluidic circuit structure disposed on the base.
The microfluidic circuit structure comprises a flow region and at least one isolation enclosure.
The isolation enclosure has a connection region, a separation region, and a displacement force generation region fluidly connected to the separation region.
The method is
Comprising the steps of: illuminating a selected discrete area adjacent to one or more micro object placed in a fluid medium in the enclosure of the microfluidic device, the selected discrete areas, said displacement force generator Steps placed in the area and
For a first period of time sufficient to heat a portion of the fluid medium, the illumination of the selected discrete region is maintained to generate one or more bubbles in the heated portion of the fluid medium. Steps to generate removal power and
Including
The one or more bubbles are at least one selected from the following (a) to (c).
(A) Persistent bubbles that displace the fluid medium surrounding the one or more micro-objects ;
(B) One or more bubbles that generate a shear flow of the fluid medium towards the one or more micro-objects; or (c) configured to flow towards the one or more micro-objects a plurality of bubbles, said plurality of at least one bubble plurality of bubbles which are configured to contact the one or more minute objects at the meniscus of the bubble,
The method is
Further Mi including the step of removing by the removal forces said one or more minute objects from the microfluidic device,
The fluid medium around the one or more micro-objects is displaced by the removing force.
Method. The method of claim 1, wherein the selected discrete region has an area of 10 square μm to 200 square μm or an area of 25 square μm to 100 square μm. The method of claim 1 or 2, wherein the illuminating step comprises illuminating the selected discrete region with a laser. The method of any one of claims 1 to 3, wherein the one or more microobjects do not move due to gravity, dielectrophoresis ( DEP ) , or perfusion in the flow region of the microfluidic device. Claims 1 to 4, further comprising maintaining the one or more micro-objects in the fluid medium in the enclosure for a second time period prior to performing the step of illuminating the selected discrete region. The method according to any one of the above. The method of any one of claims 1-5, wherein the one or more microobjects are placed and / or maintained on the surface of the base within the at least one isolation enclosure. The method according to any one of claims 1 to 6, wherein the step of illuminating the selected discrete region comprises illuminating with illumination having an incident power in the range of 1 mW to 1000 mW. The method of claim 7, wherein the first time period ranges from 10 microseconds to 3000 milliseconds or 100 milliseconds to 3 minutes. Step of illuminating said selected discrete areas, wherein the lighting base, microfluidic material of the wall, or including a directing the heat target, the method according to any one of claims 1 to 8. If the one or a plurality of micro-objects, are disposed on the at least one isolation enclosure within said enclosure,
The selected discrete region contains or contains selected points of microfluidic circuit material that help demarcate the at least one isolation enclosure.
The selected discrete region comprises a sacrificial feature that is placed within the at least one isolation enclosure.
The method according to any one of claims 1 to 9. The method of claim 9 or 10, wherein the first time period ranges from 10 microseconds to 200 milliseconds. A method of removing one or more microscopic objects within a microfluidic device.
The microfluidic device comprises an enclosure, the enclosure comprising a base and a microfluidic circuit structure disposed on the base.
The microfluidic circuit structure comprises a flow region and at least one isolation enclosure.
The isolation enclosure has a connection region, a separation region, and a displacement force generation region fluidly connected to the separation region.
The method is
Comprising the steps of: illuminating a selected discrete area adjacent to one or more micro object placed in a fluid medium in the enclosure of the microfluidic device, the selected discrete areas, said displacement force generator Steps placed in the area and
For a first period of time sufficient to heat a portion of the fluid medium, the illumination of the selected discrete region is maintained to generate one or more bubbles in the heated portion of the fluid medium. Steps to generate cavitation force and
A step of removing the one or more micro-objects from the microfluidic device with the cavitation force.
Only including,
The fluid medium around the one or more micro-objects is displaced by the cavitation force.
Method. 12. The method of claim 12, wherein the first time period is in the range of 100 ms to 3 minutes, 500 ms to 3000 ms, or 1000 ms to 2000 ms. When the one or more micro-objects are maintained within at least one isolation enclosure within the enclosure, the selected discrete region comprises at least a portion of the wall forming the tip of the isolation enclosure. , said wall, said positioned opposite to the base end opening into the flow region, wherein the one or more microscopic objects, said Ru disposed in the isolation region of the isolation enclosure of claims 1 to 13 The method according to any one item. Wherein one or more of the at least one further including the step of unloading from quarantine the enclosure a micro-material disposed within said enclosure, the method according to any one of claims 1 14. Wherein one or more of the flow region further including the step of unloading from the enclosure the minute object the microfluidic device, the method according to any one of claims 1 15. 15. The method of claim 15, wherein the step of removing the one or more micro-objects from the at least one isolation enclosure comprises moving the one or more micro-objects by dielectrophoretic force. 16. The method of claim 16, wherein the step of removing the one or more microobjects from the flow region comprises using a fluid flow. The method of claim 1, wherein the connection area of the isolation enclosure is open perpendicular to the flow area. At the base,
The microfluidic circuit structure arranged on the base and
A microfluidic device equipped with
The microfluidic circuit structure comprises a flow region and an isolation enclosure having a single opening to the flow region.
The isolation enclosure has a connection region, a separation region, and a displacement force generation region fluidly connected to the separation region.
The fluid connection is configured to prevent micro-objects from moving from the separation region to the displacement force generation region.
The connection region has a proximal opening to the flow region and a distal opening to the separation region.
The separation region has at least one fluid connection to the displacement force generation region.
The displacement force generating region further comprises a thermal target.
Microfluidic device. Claim that the at least one fluid connection between the separation region and the displacement force generation region has a cross-sectional dimension configured to prevent the microobject from moving from the separation region to the displacement force generation region. 20. The microfluidic device. The at least one fluid connection between the separation region and the displacement force generation region comprises one or more barrier modules.
The one or more barrier modules are configured to prevent the microobjects from moving from the separation region to the displacement force generation region.
The microfluidic device according to claim 20. The microfluidic circuit structure at least partially defines the isolation enclosure.
The thermal target is located in the microfluidic circuit structure.
The microfluidic device according to claim 20. The base defines the isolation enclosure at least partially.
The thermal target is located on the inner surface of the base.
The microfluidic device according to claim 20. The microfluidic device of claim 20, wherein the thermal target is a sacrificial feature. 20. The microfluidic device of claim 20, wherein the thermal target is located in a portion of the displacement force generating region at the tip to at least one fluid connection to the separation region. 9. The method of claim 9, wherein the thermal target comprises metal deposits, patterns of metal deposits, or microstructures patterned on the surface.

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